LOCUS       V044800                 9499 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V044800
VERSION     V044800
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     LTR             234..414
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /label="5' LTR (truncated)"
     misc_feature    458..583
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
                     /label="HIV-1 Ψ"
     misc_feature    1076..1309
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
                     /label="RRE"
     CDS             1493..1537
                     /codon_start=1
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /product="antigenic peptide corresponding to amino acids
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
                     et al., 2013)"
                     /transl_table=1
                     /translation="KNEQELLELDKWASL"
                     /label="gp41 peptide"
     misc_feature    1804..1920
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
                     /label="cPPT/CTS"
     enhancer        1933..2236
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        2237..2440
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     CDS             2475..4718
                     /label="KLHL1(NM_020866)"
                     /note="KLHL1(NM_020866)"
                     /gene="KLHL1"
     CDS             4725..4790
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     CDS             4800..4856
                     /codon_start=1
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /transl_table=1
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /label="P2A"
     CDS             4869..5588
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /transl_table=1
                     /translation="M,V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL
                     TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG
                     IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV
                     LLEFVTAAGITLGMDELYK*"
                     /label="EGFP"
     misc_feature    5595..6182
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             6256..6489
                     /note="self-inactivating&nbsp;3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (ΔU3)"
     polyA_signal    6561..6682
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      6701..6836
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     complement(6874..6890)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6898..6914
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     promoter        complement(6922..6952)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6967..6988
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(7276..7864)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(8035..8895)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     promoter        complement(8896..9000)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     9474..9490
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 fwd"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
      241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct
      481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
      661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
      721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
      781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
      841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
      901 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg
      961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc
     1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt
     1801 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     1861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt
     1921 atcgatacta gtcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1981 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     2041 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     2101 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     2161 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     2221 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     2281 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     2341 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     2401 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
     2461 ccgctagcgc caccatgtca ggctctgggc gaaaagactt cgatgtgaag cacattctgc
     2521 gactccgctg gaaactcttc agccacccgt ctccttccac cggcggcccg gcggggggag
     2581 gctgcctgca acaggacggc agtggcagct ttgagcactg gggacccagc cagagtcgcc
     2641 tgctcaaaag ccaagagaga agcggtgtga gcactttctg gaagaagcct tcctcctctt
     2701 cctcttcatc gtcctccccg tcctcttcct cctcttcctt caatccgctg aatggcaccc
     2761 tgcttccagt tgccacgagg ctgcagcaag gggctcctgg gcagggcact cagcagccag
     2821 ccaggactct cttctacgtg gagtcactag aggaggaggt ggtgccaggc atggactttc
     2881 ctggaccaca cgaaaaaggg ctggttctgc aagagctcaa agtggagcca gacaactcta
     2941 gccaggcaac aggtgaagga tgtggacaca ggctgtcatc aaccggccat tcaatgacac
     3001 ctcaaagtga tttggactcc agtagctctg aagaattcta tcaagctgtt catcatgctg
     3061 agcaaacctt cagaaagatg gaaagttatt tgaagcagca gcaactttgt gatgttatcc
     3121 tgattgttgg gaaccgaaag atacctgcac ataggcttgt tctgagttca gtctccgact
     3181 attttgcggc catgtttaca agtgatgttt gtgaagccaa gcaagaggag atcaaaatgg
     3241 aaggcataga ccccaatgct ctctgggacc ttgtccaatt tgcatataca ggctgcttgg
     3301 aattaaaaga ggacaccatt gagaaccttc ttgctgcagc gtgccttctt cagcttccac
     3361 aggtggtgga agtgtgctgc cacttcctca tgaagctttt gcatccatct aactgtttag
     3421 gaattcgagc cttcgcagat gctcaaggat gcattgagtt aatgaaggtg gcccacagct
     3481 acacaatgga aaacataatg gaagttatca gaaatcaaga gtttttactc cttccagctg
     3541 aggagctcca taaactactg gccagtgatg atgtcaatgt tcctgatgaa gaaaccatct
     3601 tccatgcatt gatgatgtgg gtcaagtatg acatgcagag tagatgcaat gacctgagca
     3661 tgcttcttgc ctttataaga ctgccactgc ttccaccaca gatattggct gacctagaaa
     3721 atcatgcatt atttaagaat gatctggaat gtcaaaagct gattctagaa gcaatgaaat
     3781 accatctatt gccagaaaga agaactttaa tgcaaagtcc gagaactaaa cccagaaaat
     3841 ctacagtcgg aactttgtat gctgtaggag gaatggataa caacaaagga gctacaacta
     3901 tagagaaata tgatctgaga acaaatctgt ggatccaggc agggatgatg aatggcagaa
     3961 ggctgcagtt tggtgtggct gttattgatg acaaactctt tgtaattgga ggtcgagatg
     4021 gcttaaagac attgaacact gttgaatgtt acaatcccaa aaccaagaca tggactgtct
     4081 taccaccaat gtcaacacac agacatggtc taggtgtaac agtacttgaa ggccctattt
     4141 atgctgtggg aggccatgat ggctggagct atctgaatac agtggaaagg tgggatccac
     4201 agagtcaaca atggacattt gtagccagta tgtcaattgc tcggagcaca gttggtgtag
     4261 cagcattgaa tggcaagttg tattcagttg gaggtcgtga tggaagttcc tgtttgagtt
     4321 caatggaata ttatgatcct catacaaata agtggaacat gtgtgctccc atgtgtaaga
     4381 ggagaggggg tgtcggagtg gccacatgtg acggttttct ttatgcagta ggaggtcatg
     4441 atgctcctgc ttcaaatcac tgttcccggc tactggatta tgtagaaaga tatgatccca
     4501 aaacagacac ttggaccatg gtggctcctt tgagtatgcc cagagatgct gttggggtct
     4561 gtctccttgg tgacagatta tatgctgttg gtggctatga tggacagaca tacctcaaca
     4621 ctatggaatc ctatgaccca caaactaatg agtggacaca gatggcttcc ttgaatattg
     4681 ggagagcagg tgcctgtgtg gtagtcatca agcaacctgt cgacgactac aaagaccatg
     4741 acggtgatta taaagatcat gacatcgact acaaggatga cgatgacaag ggaagcggag
     4801 ccactaactt ctccctgttg aaacaagcag gggatgtcga agagaatccc gggccagaat
     4861 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     4921 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     4981 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     5041 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     5101 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     5161 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     5221 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     5281 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     5341 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     5401 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     5461 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     5521 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     5581 acaagtaagg atccaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     5641 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     5701 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     5761 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     5821 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     5881 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     5941 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct
     6001 ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     6061 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     6121 ctcttccgcg tctccgcctt cgccctcaga cgagtcggat ctccctttgg ccgcctcccc
     6181 gcctggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     6241 aagaaaaggg gggactggaa gggctaattc actcccaacg aaaataagat ctgctttttg
     6301 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     6361 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     6421 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     6481 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     6541 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     6601 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     6661 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     6721 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     6781 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctaga
     6841 cttttgcaga gacggcccaa attcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     6901 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     6961 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     7021 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     7081 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     7141 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     7201 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     7261 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     7321 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     7381 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     7441 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     7501 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     7561 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     7621 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     7681 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     7741 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7801 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7861 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     7921 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7981 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     8041 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     8101 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     8161 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     8221 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     8281 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     8341 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     8401 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     8461 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     8521 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     8581 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     8641 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     8701 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     8761 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     8821 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     8881 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     8941 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     9001 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     9061 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     9121 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     9181 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     9241 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     9301 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     9361 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     9421 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     9481 gacggccagt gccaagctg
//
