LOCUS       V022674                10097 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V022674
VERSION     V022674
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"
     CDS             683..4744
                     /label="ROCK1(NM_005406)"
                     /note="ROCK1(NM_005406)"
                     /gene="ROCK1"
     CDS             4784..4849
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    4873..5461
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    5527..5575
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      5777..6205
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        6219..6548
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             6615..7409
                     /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    7585..7706
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(7755..7771)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    7779..7795
                     /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(7803..7833)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    7848..7869
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(8157..8745)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(8916..9776)
                     /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(9777..9881)
                     /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 gctctggcta gcgtttaaac ttaagcttgg
      661 taccgagctc ggatccgcca ccatgtcgac tggggacagt tttgagactc gatttgaaaa
      721 aatggacaac ctgctgcggg atcccaaatc ggaagtgaat tcggattgtt tgctggatgg
      781 attggatgct ttggtatatg atttggattt tcctgcctta agaaaaaaca aaaatattga
      841 caacttttta agcagatata aagacacaat aaataaaatc agagatttac gaatgaaagc
      901 tgaagattat gaagtagtga aggtgattgg tagaggtgca tttggagaag ttcaattggt
      961 aaggcataaa tccaccagga aggtatatgc tatgaagctt ctcagcaaat ttgaaatgat
     1021 aaagagatct gattctgctt ttttctggga agaaagggac atcatggctt ttgccaacag
     1081 tccttgggtt gttcagcttt tttatgcatt ccaagatgat cgttatctct acatggtgat
     1141 ggaatacatg cctggtggag atcttgtaaa cttaatgagc aactatgatg tgcctgaaaa
     1201 atgggcacga ttctatactg cagaagtagt tcttgcattg gatgcaatcc attccatggg
     1261 ttttattcac agagatgtga agcctgataa catgctgctg gataaatctg gacatttgaa
     1321 gttagcagat tttggtactt gtatgaagat gaataaggaa ggcatggtac gatgtgatac
     1381 agcggttgga acacctgatt atatttcccc tgaagtatta aaatcccaag gtggtgatgg
     1441 ttattatgga agagaatgtg actggtggtc ggttggggta tttttatacg aaatgcttgt
     1501 aggtgataca cctttttatg cagattcttt ggttggaact tacagtaaaa ttatgaacca
     1561 taaaaattca cttacctttc ctgatgataa tgacatatca aaagaagcaa aaaaccttat
     1621 ttgtgccttc cttactgaca gggaagtgag gttagggcga aatggtgtag aagaaatcaa
     1681 acgacatctc ttcttcaaaa atgaccagtg ggcttgggaa acgctccgag acactgtagc
     1741 accagttgta cccgatttaa gtagtgacat tgatactagt aattttgatg acttggaaga
     1801 agataaagga gaggaagaaa cattccctat tcctaaagct ttcgttggca atcaactacc
     1861 ttttgtagga tttacatatt atagcaatcg tagatactta tcttcagcaa atcctaatga
     1921 taacagaact agctccaatg cagataaaag cttgcaggaa agtttgcaaa aaacaatcta
     1981 taagctggaa gaacagctgc ataatgaaat gcagttaaaa gatgaaatgg agcagaagtg
     2041 cagaacctca aacataaaac tagacaagat aatgaaagaa ttggatgaag agggaaatca
     2101 aagaagaaat ctagaatcta cagtgtctca gattgagaag gagaaaatgt tgctacagca
     2161 tagaattaat gagtaccaaa gaaaagctga acaggaaaat gagaagagaa gaaatgtaga
     2221 aaatgaagtt tctacattaa aggatcagtt ggaagactta aagaaagtca gtcagaattc
     2281 acagcttgct aatgagaagc tgtcccagtt acaaaagcag ctagaagaag ccaatgactt
     2341 acttaggaca gaatcggaca cagctgtaag attgaggaag agtcacacag agatgagcaa
     2401 gtcaattagt cagttagagt ccctgaacag agagttgcaa gagagaaatc gaattttaga
     2461 gaattctaag tcacaaacag acaaagatta ttaccagctg caagctatat tagaagctga
     2521 acgaagagac agaggtcatg attctgagat gattggagac cttcaagctc gaattacatc
     2581 tttacaagag gaggtgaagc atctcaaaca taatctcgaa aaagtggaag gagaaagaaa
     2641 agaggctcaa gacatgctta atcactcaga aaaggaaaag aataatttag agatagattt
     2701 aaactacaaa cttaaatcat tacaacaacg gttagaacaa gaggtaaatg aacacaaagt
     2761 aaccaaagct cgtttaactg acaaacatca atctattgaa gaggcaaagt ctgtggcaat
     2821 gtgtgagatg gaaaaaaagc tgaaagaaga aagagaagct cgagagaagg ctgaaaatcg
     2881 ggttgttcag attgagaaac agtgttccat gctagacgtt gatctgaagc aatctcagca
     2941 gaaactagaa catttgactg gaaataaaga aaggatggag gatgaagtta agaatctaac
     3001 cctgcaactg gagcaggaat caaataagcg gctgttgtta caaaatgaat tgaagactca
     3061 agcatttgag gcagacaatt taaaaggttt agaaaagcag atgaaacagg aaataaatac
     3121 tttattggaa gcaaagagat tattagaatt tgagttagct cagcttacga aacagtatag
     3181 aggaaatgaa ggacagatgc gggagctaca agatcagctt gaagctgagc aatatttctc
     3241 gacactttat aaaacccagg taaaggaact taaagaagaa attgaagaaa aaaacagaga
     3301 aaatttaaag aaaatacagg aactacaaaa tgaaaaagaa actcttgcta ctcagttgga
     3361 tctagcagaa acaaaagctg agtctgagca gttggcgcga ggccttctgg aagaacagta
     3421 ttttgaattg acgcaagaaa gcaagaaagc tgcttcaaga aatagacaag agattacaga
     3481 taaagatcac actgttagtc ggcttgaaga agcaaacagc atgctaacca aagatattga
     3541 aatattaaga agagagaatg aagagctaac agagaaaatg aagaaggcag aggaagaata
     3601 taaactggag aaggaggagg agatcagtaa tcttaaggct gcctttgaaa agaatatcaa
     3661 cactgaacga acccttaaaa cacaggctgt taacaaattg gcagaaataa tgaatcgaaa
     3721 agattttaaa attgatagaa agaaagctaa tacacaagat ttgagaaaga aagaaaagga
     3781 aaatcgaaag ctgcaactgg aactcaacca agaaagagag aaattcaacc agatggtagt
     3841 gaaacatcag aaggaactga atgacatgca agcgcaattg gtagaagaat gtgcacatag
     3901 gaatgagctt cagatgcagt tggccagcaa agagagtgat attgagcaat tgcgtgctaa
     3961 acttttggac ctctcggatt ctacaagtgt tgctagtttt cctagtgctg atgaaactga
     4021 tggtaacctc ccagagtcaa gaattgaagg ttggctttca gtaccaaata gaggaaatat
     4081 caaacgatat ggctggaaga aacagtatgt tgtggtaagc agcaaaaaaa ttttgttcta
     4141 taatgacgaa caagataagg agcaatccaa tccatctatg gtattggaca tagataaact
     4201 gtttcacgtt agacctgtaa cccaaggaga tgtgtataga gctgaaactg aagaaattcc
     4261 taaaatattc cagatactat atgcaaatga aggtgaatgt agaaaagatg tagagatgga
     4321 accagtacaa caagctgaaa aaactaattt ccaaaatcac aaaggccatg agtttattcc
     4381 tacactctac cactttcctg ccaattgtga tgcctgtgcc aaacctctct ggcatgtttt
     4441 taagccaccc cctgccctag agtgtcgaag atgccatgtt aagtgccaca gagatcactt
     4501 agataagaaa gaggacttaa tttgtccatg taaagtaagt tatgatgtaa catcagcaag
     4561 agatatgctg ctgttagcat gttctcagga tgaacaaaaa aaatgggtaa ctcatttagt
     4621 aaagaaaatc cctaagaatc caccatctgg ttttgttcgt gcttcccctc gaacgctttc
     4681 tacaagatcc actgcaaatc agtctttccg gaaagtggtc aaaaatacat ctggaaaaac
     4741 tagtgaattc tgcagatatc cagcacagtg gcggccgctc gaggactaca aagaccatga
     4801 cggtgattat aaagatcatg acatcgacta caaggatgac gatgacaagt agtgagggcc
     4861 cgatatctcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     4921 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     4981 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     5041 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     5101 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     5161 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     5221 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt
     5281 ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     5341 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     5401 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     5461 cctggaaacg ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     5521 ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa
     5581 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     5641 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     5701 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg cagctggggc
     5761 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5821 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5881 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct
     5941 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6001 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6061 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     6121 tattcttttg atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg
     6181 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     6241 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     6301 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     6361 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     6421 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     6481 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     6541 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     6601 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     6661 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     6721 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     6781 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     6841 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     6901 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     6961 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     7021 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     7081 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     7141 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     7201 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     7261 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     7321 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     7381 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgcga aatgaccgac
     7441 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     7501 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     7561 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     7621 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     7681 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     7741 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     7801 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     7861 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     7921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     7981 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     8041 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     8101 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     8161 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     8221 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     8281 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     8341 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     8401 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     8461 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     8521 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     8581 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     8641 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     8701 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     8761 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     8821 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     8881 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     8941 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     9001 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     9061 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     9121 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     9181 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     9241 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     9301 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     9361 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     9421 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     9481 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     9541 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     9601 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     9661 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     9721 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     9781 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     9841 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     9901 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtcgact ctcagtacaa
     9961 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
    10021 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
    10081 tgaagaatct gcttagg
//
