LOCUS       V032356                 7964 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032356
VERSION     V032356
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        235..614
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        615..818
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        863..881
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             923..1639
                     /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"
     CDS             1676..3454
                     /label="Top1mt(NM_028404)"
                     /note="Top1mt(NM_028404)"
                     /gene="Top1mt"
     CDS             3461..3526
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     polyA_signal    3564..3788
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      3834..4262
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4276..4605
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             4672..5466
                     /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    5640..5761
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(5810..5826)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5834..5850
                     /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(5858..5888)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5903..5924
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6212..6797)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6968..7828)
                     /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(7829..7933)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
      961 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1021 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1081 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1141 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1201 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1261 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1321 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1381 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1441 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1501 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1561 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1621 catggacgag ctgtacaagg gcggtggagg cagtggtgga ggcggtagcg aattcatgct
     1681 gctgctgtgg ctcagagccc tgtgccggcg tttccagcat gtcccctgcc gcgtcccttc
     1741 ctgtcaggtg tcccgggtcg cgaaggcgaa cagagccggg tgggaagaaa cgagcaagag
     1801 cagtgtgaag tggaggcagc tggagcacaa gggcccgtac ttcaccccag cctacgagcc
     1861 ccttcctgac ggagtgcact tcctctacga tggcaaaccg ctgaagctga gcttggcagc
     1921 ggaggaggtc gctaccttct atgggaaaat gctgcatctt gagtgtacga gcaaggaagt
     1981 ctttcggagg aacttcttca gtgactggcg gaaggagatg acacctgagg agaggaggct
     2041 catcacacac cttgacaaat gtgacttcac ggagatccat aagcacttcc tggaaagagc
     2101 tgaggcccgc aggaccctgc ccagggagca gaagcagaag ctaaaggaag aggccgagaa
     2161 actgcagcag gagtttggct attgcgtttt agatggccac agagaaaaaa taggcaattt
     2221 caagacggaa ccacccggct tgttccgtgg ccgaggtgac catcccaaga tggggatgtt
     2281 gaagaggagg gtcatgccag aggatgtggt cattaactgc agcagggatt ccaagatccc
     2341 tgagccccca gccggtcacc agtggaaaga ggtacgctca gataacacag tcatgtggct
     2401 ggctgcctgg gtggagcaca tccagaactc cttcaagtat gtcatactga accccagctc
     2461 caagccgaag ggggagatgg attggcagaa gtatgaggta gcgcgacgct tgaagggggt
     2521 tgtggacaag atccgtgccc agtatcaggc tgactggaag tcaccagaaa tgaagaagag
     2581 acagctggct gtcgcccttt attttattga taagctggca ctgcggacag gcaatgagaa
     2641 ggaagagggc gagacggccg acaccgtggg ctgctgctcg ctccgtgtgg agcacatccg
     2701 actgcacgcg cccgcaggtg gccaggagca cgttgtagag ctggacttct tgggaaagga
     2761 ttccatccgc tataagaacc acgtgacggt ggagaagctc gtgttccaga accttcagca
     2821 cttcatggaa gacaaggacc ctagggatga cctctttgat gcactgacca cttccagcct
     2881 gaacaagcac ttgcaggact tgatggaagg gctgacagcc aaggtgttcc ggacctacaa
     2941 tgcctctgtc actctgcagg agcagctgcg ggtgctgacc cgtgcggaag acagcgtaac
     3001 ctgcaaagtc ttagcttaca accgggcgaa ccgggctgtg gcagtcctct gtaaccatca
     3061 gagagcagtt cccaagacct tcgagaagtc gatgcagaca cttcaaaaaa agattgagac
     3121 aaagaaggca caggtggctg aggcacaggt agagctgcag aaagcagaga ctgacctgag
     3181 aatgagaggg gacagcaaac ccaaaagttt ccttcagaag cagcgacgat tgttaaagct
     3241 ggaagaacag ctggcccgat tgtgcgcgaa ggccacagac aaggaggaga ataagcaggt
     3301 ggccctgggc accgccaagc tcaactatct ggaccccagg atcagcatcg cctggtgtaa
     3361 gagatttgga gtgcctgtgg agaagatcta caacaagacc cagagagaga ggtttgcctg
     3421 ggccttcaac caggcaggtg aagactttga attcctcgag gactacaaag accatgacgg
     3481 tgattataaa gatcatgaca tcgactacaa ggatgacgat gacaagtagt gagggcccgt
     3541 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3601 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3661 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3721 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3781 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3841 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3901 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3961 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4021 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4081 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4141 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4201 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4261 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     4321 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     4381 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     4441 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4501 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     4561 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     4621 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     4681 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4741 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4801 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4861 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4921 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4981 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     5041 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     5101 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     5161 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     5221 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     5281 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     5341 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     5401 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     5461 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     5521 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     5581 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     5641 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5701 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5761 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5821 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5881 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5941 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6001 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     6061 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6121 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6181 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6241 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6301 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6361 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6421 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6481 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6541 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6601 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6661 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6721 ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg caagcagcag attacgcgca
     6781 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6841 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6901 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6961 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7021 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7081 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7141 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7201 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7261 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7321 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7381 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7441 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7501 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7561 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7621 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7681 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7741 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7801 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7861 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7921 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
