LOCUS       V027722                 8580 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V027722
VERSION     V027722
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        50..429
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        430..633
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        678..696
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             756..3260
                     /label="Tlr4(NM_021297)"
                     /note="Tlr4(NM_021297)"
                     /gene="Tlr4"
     CDS             3267..3332
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    3356..3944
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    4010..4058
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      4260..4688
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4702..5031
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             5098..5892
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin®)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /transl_table=1
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
                     /label="NeoR/KanR"
     polyA_signal    6068..6189
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(6238..6254)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6262..6278
                     /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(6286..6316)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6331..6352
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6640..7228)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7399..8259)
                     /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(8260..8364)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      661 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta
      721 aacttaagct tggtaccgag ctcgaattcg ccaccatgat gcctccctgg ctcctggcta
      781 ggactctgat catggcactg ttcttctcct gcctgacacc aggaagcttg aatccctgca
      841 tagaggtagt tcctaatatt acctaccaat gcatggatca gaaactcagc aaagtccctg
      901 atgacattcc ttcttcaacc aagaacatag atctgagctt caaccccttg aagatcttaa
      961 aaagctatag cttctccaat ttttcagaac ttcagtggct ggatttatcc aggtgtgaaa
     1021 ttgaaacaat tgaagacaag gcatggcatg gcttacacca cctctcaaac ttgatactga
     1081 caggaaaccc tatccagagt ttttccccag gaagtttctc tggactaaca agtttagaga
     1141 atctggtggc tgtggagaca aaattggcct ctctagaaag cttccctatt ggacagctta
     1201 taaccttaaa gaaactcaat gtggctcaca attttataca ttcctgtaag ttacctgcat
     1261 atttttccaa tctgacgaac ctagtacatg tggatctttc ttataactat attcaaacta
     1321 ttactgtcaa cgacttacag tttctacgtg aaaatccaca agtcaatctc tctttagaca
     1381 tgtctttgaa cccaattgac ttcattcaag accaagcctt tcagggaatt aagctccatg
     1441 aactgactct aagaggtaat tttaatagct caaatataat gaaaacttgc cttcaaaacc
     1501 tggctggttt acacgtccat cggttgatct tgggagaatt taaagatgaa aggaatctgg
     1561 aaatttttga accctctatc atggaaggac tatgtgatgt gaccattgat gagttcaggt
     1621 taacatatac aaatgatttt tcagatgata ttgttaagtt ccattgcttg gcgaatgttt
     1681 ctgcaatgtc tctggcaggt gtatctataa aatatctaga agatgttcct aaacatttca
     1741 aatggcaatc cttatcaatc attagatgtc aacttaagca gtttccaact ctggatctac
     1801 cctttcttaa aagtttgact ttaactatga acaaagggtc tatcagtttt aaaaaagtgg
     1861 ccctaccaag tctcagctat ctagatctta gtagaaatgc actgagcttt agtggttgct
     1921 gttcttattc tgatttggga acaaacagcc tgagacactt agacctcagc ttcaatggtg
     1981 ccatcattat gagtgccaat ttcatgggtc tagaagagct gcagcacctg gattttcagc
     2041 actctacttt aaaaagggtc acagaattct cagcgttctt atcccttgaa aagctacttt
     2101 accttgacat ctcttatact aacaccaaaa ttgacttcga tggtatattt cttggcttga
     2161 ccagtctcaa cacattaaaa atggctggca attctttcaa agacaacacc ctttcaaatg
     2221 tctttgcaaa cacaacaaac ttgacattcc tggatctttc taaatgtcaa ttggaacaaa
     2281 tatcttgggg ggtatttgac accctccata gacttcaatt attaaatatg agtcacaaca
     2341 atctattgtt tttggattca tcccattata accagctgta ttccctcagc actcttgatt
     2401 gcagtttcaa tcgcatagag acatctaaag gaatactgca acattttcca aagagtctag
     2461 ccttcttcaa tcttactaac aattctgttg cttgtatatg tgaacatcag aaattcctgc
     2521 agtgggtcaa ggaacagaag cagttcttgg tgaatgttga acaaatgaca tgtgcaacac
     2581 ctgtagagat gaatacctcc ttagtgttgg attttaataa ttctacctgt tatatgtaca
     2641 agacaatcat cagtgtgtca gtggtcagtg tgattgtggt atccactgta gcatttctga
     2701 tataccactt ctattttcac ctgatactta ttgctggctg taaaaagtac agcagaggag
     2761 aaagcatcta tgatgcattt gtgatctact cgagtcagaa tgaggactgg gtgagaaatg
     2821 agctggtaaa gaatttagaa gaaggagtgc cccgctttca cctctgcctt cactacagag
     2881 actttattcc tggtgtagcc attgctgcca acatcatcca ggaaggcttc cacaagagcc
     2941 ggaaggttat tgtggtagtg tctagacact ttattcagag ccgttggtgt atctttgaat
     3001 atgagattgc tcaaacatgg cagtttctga gcagccgctc tggcatcatc ttcattgtcc
     3061 ttgagaaggt tgagaagtcc ctgctgaggc agcaggtgga attgtatcgc cttcttagca
     3121 gaaacaccta cctggaatgg gaggacaatc ctctggggag gcacatcttc tggagaagac
     3181 ttaaaaatgc cctattggat ggaaaagcct cgaatcctga gcaaacagca gaggaagaac
     3241 aagaaacggc aacttggacc ggatccgact acaaagacca tgacggtgat tataaagatc
     3301 atgacatcga ctacaaggat gacgatgaca agtagtgagg gcccgatatc tcgacaatca
     3361 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3421 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     3481 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     3541 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     3601 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     3661 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3721 cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg
     3781 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3841 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3901 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggaa acgggggagg
     3961 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     4021 agacagaata aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     4081 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     4141 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     4201 cggggcggca ggccctgcca tagcagatct gcgcagctgg ggctctaggg ggtatcccca
     4261 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4321 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4381 gttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag
     4441 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4501 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4561 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4621 agggattttg gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4681 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4741 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4801 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4861 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4921 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     4981 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     5041 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     5101 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     5161 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     5221 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     5281 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     5341 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     5401 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     5461 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     5521 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5581 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     5641 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     5701 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5761 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     5821 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5881 gagttcttct gagcgggact ctggggttcg cgaaatgacc gaccaagcga cgcccaacct
     5941 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     6001 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     6061 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     6121 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     6181 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     6241 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     6301 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     6361 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6421 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     6481 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6541 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6601 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6661 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6721 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6781 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     6841 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6901 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6961 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     7021 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     7081 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     7141 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     7201 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     7261 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     7321 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     7381 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     7441 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     7501 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7561 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7621 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7681 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7741 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7801 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7861 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7921 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7981 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     8041 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     8101 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     8161 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     8221 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     8281 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     8341 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     8401 atcgggagat ctcccgatcc cctatggtcg actctcagta caatctgctc tgatgccgca
     8461 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     8521 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
//
