LOCUS       V045149                10536 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V045149
VERSION     V045149
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..5216
                     /label="Col2a1(NM_031163)"
                     /note="Col2a1(NM_031163)"
                     /gene="Col2a1"
     CDS             5223..5288
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    5312..5900
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    5966..6014
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      6216..6644
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        6658..6987
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             7054..7848
                     /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    8024..8145
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(8194..8210)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    8218..8234
                     /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(8242..8272)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    8287..8308
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(8596..9184)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(9355..10215)
                     /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(10216..10320)
                     /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 ccgcctcggg gctccccagt
      781 cgctggtgct gctgacgctg ctcatcgccg cggtcctacg gtgtcagggc caggatgccc
      841 aggaggctgg cagctgtctg cagaatgggc agaggtataa agataaggat gtatggaagc
      901 cctcatcttg ccgcatctgt gtgtgtgaca ctgggaatgt cctctgcgat gacattatct
      961 gtgaagaccc agactgcctc aaccccgaga tccccttcgg agagtgctgt cccatctgcc
     1021 cagctgacct cgccactgcc agtggaaaat tagggccaaa ggggcagaaa ggagaacctg
     1081 gagatatcag agatatcata ggacccagag gacctcctgg ccctcaggga cctgcaggtg
     1141 aacaaggacc cagaggtgat cgtggtgaca agggagaaaa gggtgcgcct ggaccccgtg
     1201 gcagagatgg agaacctggt acccctggaa atcctggccc cgctggccct ccaggtcccc
     1261 ctggtccccc tggccttagt gcaggaaact tcgcggctca gatggctgga gggtatgacg
     1321 agaaggctgg tggtgcccag atgggagtca tgcaagggcc catgggcccc atgggacccc
     1381 gtggaccccc aggccctgcc ggtgcccccg gccctcaagg atttcaaggc aatcctggtg
     1441 aacctggcga gcctggtgtc tctggtccca tgggtccccg aggtcctcct ggccctgctg
     1501 gaaaacctgg tgacgacggt gaagctggga agcccggaaa gtctggggaa agaggcctcc
     1561 ctggccctca gggtgctcgt ggattcccag gaaccccggg tctccccggt gtcaagggtc
     1621 acagaggtta cccaggcctc gacggtgcta agggggaagc tggtgctccg ggtgtgaagg
     1681 gtgagagtgg ttcccctggt gagaacggat ccccgggccc aatgggtccc cgtggcctgc
     1741 ctggtgagag aggacggact ggccctgctg gtgctgctgg tgctcggggt aacgatggcc
     1801 agccaggccc cgctggacct ccgggtcctg tgggtcccgc aggtggtcct ggcttccctg
     1861 gtgctcctgg tgccaagggc gaagctggtc ccactggtgc tcgcggtcct gaaggtgctc
     1921 aaggttctcg tggcgagcct ggcaatcctg ggtcccctgg gcctgcaggt gcttctggta
     1981 acccagggac tgatggtatt cctggagcca aaggatccgc tggtgctcct ggaattgctg
     2041 gtgcccctgg cttccctggg ccccgtggcc ctcccggtcc tcaaggtgca actggtcccc
     2101 ttggccccaa aggtcaggcg ggtgaacctg gcattgctgg ctttaaaggt gatcaaggcc
     2161 ccaagggaga gactggacct gctgggcccc aaggagcccc tggccccgct ggtgaagaag
     2221 gcaaacgagg tgctcgagga gagccgggtg gtgctggacc aatcggaccc cctggagaga
     2281 gaggtgctcc tggcaaccgt ggattcccag gtcaagatgg tctggcaggt cccaagggtg
     2341 cccctggaga gcgagggccc agtggcttga ctggtcccaa gggagccaac ggtgacccgg
     2401 gtcgtcctgg agaacctggt cttcctggag ccaggggtct taccggtcgc cctggtgacg
     2461 ctggtcctca aggcaaagtt ggtccttctg gagcccctgg tgaagacggt cgccctggac
     2521 ctcctggtcc tcagggagct cgtgggcagc ctggcgtcat gggtttccct ggccccaaag
     2581 gtgccaacgg cgagcctggc aaagctggtg agaagggtct ggctggcgct cctggtctga
     2641 gaggtcttcc tggaaaagac ggtgagacgg gagccgcagg accccccggc cccagtggac
     2701 ctgctggtga acgaggcgag cagggcgctc ctggaccatc agggttccag ggacttcctg
     2761 gccctcccgg tcccccaggt gaaggtggaa agcaaggtga ccagggtatt cctggtgaag
     2821 ctggagctcc tggccttgtg ggtcctcggg gcgagcgagg tttcccaggt gaacgtggct
     2881 ctcctggtgc tcagggcctt cagggtcccc gaggcctccc tggcactcct ggtactgatg
     2941 gtcccaaagg tgcagctggc ccagatggcc cccctggggc tcaggggcct ccaggtctac
     3001 agggaatgcc tggtgagaga ggagccgctg gcattgctgg gcccaaggga gacagaggcg
     3061 atgttggcga gaaaggccca gagggagctc ctgggaagga tggcggccga ggtctgactg
     3121 ggcccatcgg acccccaggc ccagcagggg ccaacggcga gaagggagaa gtcggacctc
     3181 ctggcccgtc aggaagtacc ggagctcgag gtgccccggg tgaacgcgga gagaccgggc
     3241 cacctggacc tgctggattc gctggccctc ctggtgctga tggccagcct ggtgccaagg
     3301 gtgatcaagg agaagccgga cagaaaggag atgctggtgc ccccggccca caaggcccct
     3361 cgggagcccc tgggccacag ggtcctactg gagtgactgg tcctaaggga gcccgaggtg
     3421 cccaaggtcc cccgggagcc accggattcc ctggagctgc tggccgagtt ggacccccag
     3481 gtgctaatgg caatcctgga cccgccggtc cccctggtcc tgctggaaaa gatggtccca
     3541 aaggtgttcg aggagacagt ggcccccctg gcagagctgg tgaccccggt cttcaaggtc
     3601 ctgcaggagc tcctggcgag aaaggagaac ctggagatga tggtccctct ggtcttgatg
     3661 gtcctccagg tccccagggg ctggctggtc aaaggggcat tgttggtctg cctggtcagc
     3721 gtggtgagag aggattcccc ggccttcccg gcccatcggg tgagcccggc aagcagggtg
     3781 cacctggcgc gtctggagac agaggtcctc ctggtcctgt ggggcctcct ggcctgacag
     3841 ggcctgcagg tgaacctgga cgagagggca gccctggtgc tgatggaccc cctggaagag
     3901 atggtgcagc tggagtcaag ggagatcgtg gtgagactgg agcactgggt gcccctggag
     3961 ctcctgggcc cccaggctct cctggtcctg ctggcccaac tggcaaacaa ggagacagag
     4021 gagaggctgg tgcacaaggt cctatgggtc cctcaggacc tgctggagcc cgtgggattg
     4081 caggccctca aggcccccga ggtgacaaag gagaatctgg agagcagggc gagaggggac
     4141 tgaagggaca ccgaggtttc actggactgc agggtctgcc tggccctccg ggtccttctg
     4201 gagatcaggg tgcttctggc cctgctggtc cttctggccc tagaggtcca cctggccctg
     4261 ttggtccctc tggcaaagat ggctctaatg gaatccctgg ccccatcggg cctccaggtc
     4321 cccgtggacg ctcaggagaa acaggccctg ttggtccccc tggaagtccc ggtcctcctg
     4381 gccctccagg tcctcctggt cctggcatcg acatgtcagc ctttgctggc ttagggcaga
     4441 gagagaaggg ccccgacccc atgcagtaca tgcgggccga cgaggcagac agtaccttga
     4501 gacagcacga cgtggaggtg gacgctacac tcaagtcact gaacaaccag attgagagca
     4561 tccgcagccc cgacggctcc cgcaagaacc ctgctcgcac ttgccaagac ctgaaactct
     4621 gccaccccga gtggaagagc ggagactact ggattgatcc caaccagggc tgcaccttgg
     4681 acgccatgaa agttttctgc aacatggaga ccggcgagac ttgcgtctac cccaacccag
     4741 cgactgtccc tcggaaaaac tggtggagca gcaagagcaa ggaaaagaaa cacatctggt
     4801 ttggagagac catgaacggt ggcttccact tcagctatgg cgatggcaac ctggctccca
     4861 acaccgctaa cgtccagatg actttcctcc gtctactgtc cactgagggc tcccagaaca
     4921 tcacctacca ctgtaagaac agcatcgcct acctggacga agcggctggc aacctcaaga
     4981 aggccttgct catccagggc tccaatgatg tagagatgag ggccgagggc aacagcaggt
     5041 tcacatacac tgccctgaag gatggctgca cgaaacacac tggtaagtgg ggcaagaccg
     5101 tcatcgagta ccgatcacag aagacctccc gccttcccat tattgacatc gcacccatgg
     5161 acattggagg ggctgaacag gaatttggtg tggacatagg gcctgtctgc ttcttgggat
     5221 ccgactacaa agaccatgac ggtgattata aagatcatga catcgactac aaggatgacg
     5281 atgacaagta gtgagggccc gatatctcga caatcaacct ctggattaca aaatttgtga
     5341 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     5401 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     5461 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     5521 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     5581 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     5641 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     5701 ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     5761 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     5821 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     5881 cctttgggcc gcctccccgc ctggaaacgg gggaggctaa ctgaaacacg gaaggagaca
     5941 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt
     6001 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     6061 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     6121 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     6181 agatctgcgc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     6241 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     6301 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     6361 tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     6421 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     6481 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     6541 actcaaccct atctcggtct attcttttga tttataaggg attttgggga tttcggccta
     6601 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     6661 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     6721 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     6781 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     6841 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     6901 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     6961 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     7021 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     7081 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     7141 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     7201 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     7261 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     7321 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     7381 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     7441 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     7501 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     7561 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     7621 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     7681 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     7741 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     7801 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     7861 ggttcgcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     7921 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     7981 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     8041 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     8101 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     8161 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     8221 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     8281 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     8341 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     8401 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     8461 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     8521 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     8581 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     8641 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     8701 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     8761 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     8821 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     8881 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     8941 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     9001 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     9061 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     9121 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     9181 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     9241 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     9301 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     9361 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     9421 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     9481 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     9541 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     9601 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     9661 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     9721 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     9781 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     9841 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     9901 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     9961 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
    10021 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
    10081 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
    10141 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
    10201 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
    10261 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
    10321 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
    10381 tggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
    10441 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
    10501 ggcttgaccg acaattgcat gaagaatctg cttagg
//
