LOCUS       V044889                 8977 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V044889
VERSION     V044889
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..4196
                     /label="KLHL18(NM_025010)"
                     /note="KLHL18(NM_025010)"
                     /gene="KLHL18"
     CDS             4203..4268
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     CDS             4278..4334
                     /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             4347..5066
                     /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    5073..5660
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             5734..5967
                     /note="self-inactivating&nbsp;3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (ΔU3)"
     polyA_signal    6039..6160
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      6179..6314
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     complement(6352..6368)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6376..6392
                     /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(6400..6430)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6445..6466
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6754..7342)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7513..8373)
                     /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(8374..8478)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     8952..8968
                     /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 caccatggtg gaggacggcg cggaggagct ggaggatctg gtgcacttct
     2521 ccgtgtctga gttgcctagt cgcggctacg gcgtcatgga ggagatccgg cggcagggca
     2581 agctgtgcga cgtgaccctc aagattgggg accacaaatt cagtgcccac cggattgtct
     2641 tagcagcctc gatcccgtat ttccatgcta tgtttacaaa tgacatgatg gagtgcaagc
     2701 aggatgagat tgtaatgcaa ggaatggacc caagtgccct ggaggctctg atcaactttg
     2761 cctacaacgg caaccttgcc attgaccagc aaaatgtcca gtcattgctg atgggggcga
     2821 gcttcctgca gctgcagagc atcaaagacg cctgctgcac attccttcga gaacggcttc
     2881 acccaaaaaa ctgcctgggt gtgcgccagt ttgctgagac aatgatgtgt gctgtgctgt
     2941 acgacgctgc caacagcttc atccaccagc actttgtgga ggtgtccatg tcagaagagt
     3001 tcctggccct gcccttggaa gacgtgcttg agctggtgtc tcgggatgag ctgaatgtca
     3061 aatctgagga gcaggtcttt gaagctgcat tggcctgggt cagatacgac cgggagcaga
     3121 ggggtcccta cctgcctgag ctgctgtcca atatccgcct gcccctctgt cggccccagt
     3181 tcctttcaga cagagtacag caggatgacc tggtgcgttg ctgccacaaa tgcagggacc
     3241 tggtagacga agcaaaggac taccacctca tgccagagcg ccggccccac ctgccagctt
     3301 tcagaacccg gccacgctgc tgcacatcca tcgctggact tatctacgct gtagggggcc
     3361 tcaactcagc aggtgattcc ctgaatgtgg tggaagtgtt cgaccccatt gccaattgct
     3421 gggagagatg ccgtcccatg acaacagccc gcagccgcgt tggcgtggct gtggtgaacg
     3481 ggcttctcta tgccatcgga ggatatgacg gccagctacg gctgagcact gtggaggcct
     3541 acaacccgga gacagacaca tggaccagag tggggagcat gaatagcaag agaagtgcca
     3601 tggggacagt cgtgctggat gggcagatct acgtctgtgg gggctacgat ggcaactctt
     3661 ccctcagctc cgtggagacc tactcacctg agacggacaa atggacagtg gtgacctcga
     3721 tgagctcgaa tcgcagtgct gctggggtta cagtctttga gggcaggata tatgtgtcag
     3781 gcggccatga tggtttgcag atcttcagca gtgtggaaca ctacaaccac cacacagcca
     3841 cctggcaccc tgcagctggc atgctcaaca agcgctgccg gcacggagcc gcctccctgg
     3901 ggagcaagat gtttgtctgc gggggctacg atggctctgg cttcctcagc attgccgaga
     3961 tgtacagctc tgtggcagac cagtggtgcc tgattgtccc catgcacacg cgcaggagcc
     4021 gggtctccct ggtggccagc tgtgggcgcc tctacgctgt tgggggctac gacggacagt
     4081 caaacctaag ctcagtggag atgtatgacc cagagacaga ctgctggaca ttcatggccc
     4141 ccatggcgtg ccatgaggga ggggtcggtg tgggctgcat ccctctcctc accatcgtcg
     4201 acgactacaa agaccatgac ggtgattata aagatcatga catcgactac aaggatgacg
     4261 atgacaaggg aagcggagcc actaacttct ccctgttgaa acaagcaggg gatgtcgaag
     4321 agaatcccgg gccagaattc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     4381 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     4441 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     4501 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     4561 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     4621 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     4681 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     4741 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     4801 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     4861 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     4921 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     4981 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     5041 tcggcatgga cgagctgtac aagtaaggat ccaatcaacc tctggattac aaaatttgtg
     5101 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     5161 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     5221 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     5281 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     5341 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     5401 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     5461 cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg
     5521 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     5581 tgctgccggc tctgcggcct cttccgcgtc tccgccttcg ccctcagacg agtcggatct
     5641 ccctttggcc gcctccccgc ctggtacctt taagaccaat gacttacaag gcagctgtag
     5701 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     5761 aataagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg
     5821 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     5881 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     5941 tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt
     6001 tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata
     6061 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     6121 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat
     6181 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     6241 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     6301 cttttttgga ggcctagact tttgcagaga cggcccaaat tcgtaatcat ggtcatagct
     6361 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6421 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6481 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6541 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6601 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6661 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6721 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6781 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6841 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6901 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6961 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7021 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7081 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7141 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     7201 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7261 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7321 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7381 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7441 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7501 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7561 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7621 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7681 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7741 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7801 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7861 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7921 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7981 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     8041 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     8101 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     8161 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     8221 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8281 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     8341 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8401 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     8461 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     8521 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     8581 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     8641 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     8701 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     8761 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc
     8821 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     8881 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     8941 gtcacgacgt tgtaaaacga cggccagtgc caagctg
//
