LOCUS       V045360                 7057 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V045360
VERSION     V045360
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"
     regulatory      913..922
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             922..987
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     CDS             991..1839
                     /label="VDAC1(NM_003374)"
                     /note="VDAC1(NM_003374)"
                     /gene="VDAC1"
     CDS             1870..2580
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /transl_table=1
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK*"
                     /label="mNeonGreen"
     polyA_signal    2657..2881
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      2927..3355
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        3369..3698
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             3765..4559
                     /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    4733..4854
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(4903..4919)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    4927..4943
                     /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(4951..4981)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    4996..5017
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(5305..5890)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6061..6921)
                     /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(6922..7026)
                     /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 aagccaccat ggactacaaa gaccatgacg gtgattataa agatcatgac
      961 atcgactaca aggatgacga tgacaagctt atggctgtgc cacccacgta tgccgatctt
     1021 ggcaaatctg ccagggatgt cttcaccaag ggctatggat ttggcttaat aaagcttgat
     1081 ttgaaaacaa aatctgagaa tggattggaa tttacaagct caggctcagc caacactgag
     1141 accaccaaag tgacgggcag tctggaaacc aagtacagat ggactgagta cggcctgacg
     1201 tttacagaga aatggaatac cgacaataca ctaggcaccg agattactgt ggaagatcag
     1261 cttgcacgtg gactgaagct gaccttcgat tcatccttct cacctaacac tgggaaaaaa
     1321 aatgctaaaa tcaagacagg gtacaagcgg gagcacatta acctgggctg cgacatggat
     1381 ttcgacattg ctgggccttc catccggggt gctctggtgc taggttacga gggctggctg
     1441 gccggctacc agatgaattt tgagactgca aaatcccgag tgacccagag caactttgca
     1501 gttggctaca agactgatga attccagctt cacactaatg tgaatgacgg gacagagttt
     1561 ggcggctcca tttaccagaa agtgaacaag aagttggaga ccgctgtcaa tcttgcctgg
     1621 acagcaggaa acagtaacac gcgcttcgga atagcagcca agtatcagat tgaccctgac
     1681 gcctgcttct cggctaaagt gaacaactcc agcctgatag gtttaggata cactcagact
     1741 ctaaagccag gtattaaact gacactgtca gctcttctgg atggcaagaa cgtcaatgct
     1801 ggtggccaca agcttggtct aggactggaa tttcaagcag gtggaggcgg atcaggaggc
     1861 ggtggatcta tggtgagcaa gggcgaggag gataacatgg cctctctccc agcgacacat
     1921 gagttacaca tctttggctc catcaacggt gtggactttg acatggtggg tcagggcacc
     1981 ggcaatccaa atgatggtta tgaggagtta aacctgaagt ccaccaaggg tgacctccag
     2041 ttctccccct ggattctggt ccctcatatc gggtatggct tccatcagta cctgccctac
     2101 cctgacggga tgtcgccttt ccaggccgcc atggtagatg gctccggata ccaagtccat
     2161 cgcacaatgc agtttgaaga tggtgcctcc cttactgtta actaccgcta cacctacgag
     2221 ggaagccaca tcaaaggaga ggcccaggtg aaggggactg gtttccctgc tgacggtcct
     2281 gtgatgacca actcgctgac cgctgcggac tggtgcaggt cgaagaagac ttaccccaac
     2341 gacaaaacca tcatcagtac ctttaagtgg agttacacca ctggaaatgg caagcgctac
     2401 cggagcactg cgcggaccac ctacaccttt gccaagccaa tggcggctaa ctatctgaag
     2461 aaccagccga tgtacgtgtt ccgtaagacg gagctcaagc actccaagac cgagctcaac
     2521 ttcaaggagt ggcaaaaggc ctttaccgat gtgatgggca tggacgagct gtacaagtaa
     2581 gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc
     2641 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2701 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2761 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2821 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2881 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     2941 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3001 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3061 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3121 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3181 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3241 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3301 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3361 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3421 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3481 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3541 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3601 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3661 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3721 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3781 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3841 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     3901 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     3961 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4021 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4081 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4141 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4201 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4261 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4321 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4381 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4441 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4501 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4561 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4621 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4681 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4741 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4801 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4861 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4921 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4981 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5041 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5101 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5161 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5221 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5281 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5341 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5401 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5461 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5521 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5581 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5641 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5701 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5761 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5821 acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5881 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5941 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6001 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6061 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6121 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6181 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6241 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6301 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6361 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6421 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6481 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6541 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6601 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6661 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6721 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6781 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6841 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6901 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6961 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7021 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
