LOCUS       V046329                10656 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V046329
VERSION     V046329
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..5336
                     /label="Col18a1(NM_001109991)"
                     /note="Col18a1(NM_001109991)"
                     /gene="Col18a1"
     CDS             5343..5408
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    5432..6020
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    6086..6134
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      6336..6764
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        6778..7107
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             7174..7968
                     /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    8144..8265
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(8314..8330)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    8338..8354
                     /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(8362..8392)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    8407..8428
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(8716..9304)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(9475..10335)
                     /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(10336..10440)
                     /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 ccaccatggc tcccgacccc agcagacgcc
      781 tctgcctgct gctgctgttg ctgctctcct gccgccttgt gcctgccagc gctgatggga
      841 attccctgag tcccctgaat cccctggtgt ggctctggcc gcccaagacc tctgattctt
      901 tggaaggccc ggtctcgaaa ccccagaaca gttcacctgt gcagtccaca gagaacccca
      961 ccacgcacgt ggtcccccag gatggcctca cagaacaaca gacgactcct gccagctccg
     1021 agctgccccc ggaggaagag gaggaggagg atcagaaagc tggccagggt ggcagccctg
     1081 caacccctgc tgtccccatt cccctggtgg cccctgctgc gagcccagac atgaaggagg
     1141 agaatgttgc gggcgttgga gccaagattc tgaatgtggc tcagggcatt cggagcttcg
     1201 tccagctgtg ggatgaggac tcgactattg ggcactctgc tgggactgag gttccagatt
     1261 cttcaatccc tacagtcctc ccctctcctg ccgagctctc cagtgctccc caggggagta
     1321 agaccactct ctggctaagc agtgccattc caagttctcc agatgctcag acaactgagg
     1381 ctggcacgct ggctgtgccc acccagctgc ctcccttcca gagcaacctc caggcaccac
     1441 tgggaagacc ctcagcccca ccagacttcc cagagaatgt tgctgaggag gtggggctgc
     1501 tgcagctcct tggagacccc ctacctgaga agatctcaca aatcgatgac cctcacgtcg
     1561 ggccggccta catctttgga ccagactcca acagtggcca ggtggcccag tatcatttcc
     1621 caaaactctt cttccgggac ttttcgctgc tgtttcatgt ccggccagcc acagaggcag
     1681 caggggtgct atttgccatc acagatgctg cccaggtggt agtctcactg ggcgtgaagc
     1741 tctcagaggt ccgagatgga cagcaaaaca tctcattgct ctacacggag cctggggcca
     1801 gccagaccca gacgggagcc agcttccgcc tacctgcatt tgttgggcag tggacacact
     1861 tcgcgctcag cgtcgacgga ggctctgtgg ctctctacgt agactgtgaa gaattccaga
     1921 gggtgccatt tgctcgggcc tcgcagggac tggagctaga gcgtggcgct ggcctctttg
     1981 tgggtcaggc tggaacagca gaccctgaca agttccaggg gatgatctca gagctgaagg
     2041 tacgcaaaac cccccgggtg agccctgtgc actgtctgga tgaagaagat gatgatgaag
     2101 accgggcatc tggagatttt ggaagtggct ttgaagaaag cagcaagtca cacaaggagg
     2161 atacatctct actacctggg ctccctcagc cacctcctgt cacttcccca cccctggctg
     2221 gaggcagcac cacagaagat cctagaacag aagaaacgga ggaagacgcc gcggtagatt
     2281 ctataggagc tgagaccctt cctggcacag gttcaagcgg tgcatgggat gaggctatcc
     2341 agaaccccgg aaggggcttg ataaagggag gtatgaaagg acaaaaggga gaaccaggtg
     2401 cccagggccc acctggccca gctgggcccc agggtcctgc cggtccagtg gtccagagcc
     2461 ccaactcaca acctgtccct ggagcacaag gacccccggg acctcagggg ccaccaggga
     2521 aggatggcac tccaggaagg gatggtgaac cgggtgaccc tggtgaagat gggagaccgg
     2581 gtgacactgg acctcaaggc tttccaggga ccccaggaga tgtgggccct aagggcgaga
     2641 agggagatcc tggtattggg ccccgaggac ctccagggcc tccagggcca ccaggaccct
     2701 ccttcagaca agacaagctg accttcattg acatggaggg atccggtttc agcggagaca
     2761 tagagagcct tagaggccca cgaggcttcc ctggcccccc ggggccccct ggtgtcccag
     2821 gacttcctgg tgagccagga cgctttggga tcaatggttc ctatgcacca ggacctgcag
     2881 gccttcctgg tgtacctggg aaggaaggac cccccggttt tccaggtccc ccgggacctc
     2941 caggtcctcc aggcaaagag ggcccaccag gagtggccgg ccagaaaggc agtgttggtg
     3001 atgtgggcat cccaggaccc aaggggagca aaggagacct tgggcccatc ggtatgcctg
     3061 gcaagtctgg cttggctgga tcccctgggc cagttggacc cccaggacct ccagggcctc
     3121 cagggccacc aggaccagga tttgctgctg gattcgatga tatggaaggc tctggaatac
     3181 ccctctggac aacagcccga agctctgatg ggctgcaggg acctcccggg tcgccgggac
     3241 tcaaggggga tcctggagtg gcaggcctac ctggagccaa gggagaagtt ggagcagatg
     3301 gagcccaggg catccctggt cccccaggaa gagaaggtgc agctggatct ccggggccaa
     3361 aaggagagaa agggatgccg ggagaaaagg gaaacccagg aaaagatgga gtgggccggc
     3421 cgggcctccc tgggcctcca ggacctccag ggcctgtgat ctatgtgtca agtgaggata
     3481 aagcaatagt gagcacgcca ggacctgagg gcaagccagg gtacgcaggc tttcctggac
     3541 ctgctggacc gaagggtgac ctgggttcca aaggcgagca gggtcttccg gggcccaagg
     3601 gtgagaaggg agagccaggc actatcttta gtcctgatgg cagagctctg ggccatcccc
     3661 agaagggagc caagggagag ccaggctttc gaggaccccc gggtccttat ggacgacctg
     3721 ggcacaaggg tgaaattggc ttccctggac ggccgggtcg acctggaacg aatggcttaa
     3781 agggagagaa aggagagcct ggagatgcca gccttgggtt cagcatgagg ggattgcctg
     3841 gcccccctgg gcctccagga cccccaggtc ctcctgggat gcccatctat gacagcaatg
     3901 catttgtgga gtctggccga cctggactac caggacagca gggtgtgcag gggccttcag
     3961 gaccaaaggg tgacaaagga gaggtgggcc cacctgggcc accagggcaa ttccccattg
     4021 acctcttcca cctggaagcg gaaatgaagg gggacaaggg agaccgaggg gatgctggac
     4081 agaaaggaga gaggggagaa cctggggctc ctggtggtgg attcttcagc tcaagtgtac
     4141 ctggcccccc cggcccacct ggataccctg gaattccggg tccaaaggga gagagcatcc
     4201 gggggccacc tggccctcct ggcccgcagg gacctcctgg cattggctat gagggtcgcc
     4261 agggtccccc aggacctccg ggacctccag gacctccctc cttccctggc cctcacagac
     4321 agactgtcag tgttcctggt cctccgggcc cacctggtcc tccaggtccc ccaggagcca
     4381 tgggtgcctc tgctgggcag gtgaggatct gggccacata ccagaccatg ctggacaaga
     4441 tccgggaggt gccggagggc tggctcatct ttgtggccga gagggaagag ctctatgtac
     4501 gcgttagaaa tggcttccgg aaggtgctgc tggaggcccg gacagccctc ccgagaggca
     4561 cgggcaatga ggtggctgct ttgcagcccc cattggtcca gcttcatgag ggcagtccat
     4621 acacccggag ggagtactcc tattccacgg cacgaccctg gcgagcagat gacatcctgg
     4681 ccaacccacc gcgcctgcca gaccgccagc cttaccccgg agttccacat caccacagtt
     4741 cctatgtgca cctgccgcca gcccgcccca ccctctcact tgctcatact catcaggact
     4801 ttcagccagt gctccacctg gtggcactga acacccccct gtctggaggc atgcgtggta
     4861 tccgtggagc agatttccag tgcttccagc aagcccgagc cgtggggctg tcgggcacct
     4921 tccgggcttt cctgtcctct aggctgcagg atctctatag catcgtgcgc cgtgctgacc
     4981 gggggtctgt gcccatcgtc aacctgaagg acgaggtgct atctcccagc tgggactccc
     5041 tgttttctgg ctcccagggt caactgcaac ccggggcccg catcttttct tttgacggca
     5101 gagatgtcct gagacaccca gcctggccgc agaagagcgt atggcacggc tcggacccca
     5161 gtgggcggag gctgatggag agttactgtg agacatggcg aactgaaact actggggcta
     5221 caggtcaggc ctcctccctg ctgtcaggca ggctcctgga acagaaagct gcgagctgcc
     5281 acaacagcta catcgtcctg tgcattgaga atagcttcat gacctctttc tccaaaggat
     5341 ccgactacaa agaccatgac ggtgattata aagatcatga catcgactac aaggatgacg
     5401 atgacaagta gtgagggccc gatatctcga caatcaacct ctggattaca aaatttgtga
     5461 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     5521 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     5581 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     5641 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     5701 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     5761 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     5821 ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     5881 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     5941 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     6001 cctttgggcc gcctccccgc ctggaaacgg gggaggctaa ctgaaacacg gaaggagaca
     6061 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt
     6121 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     6181 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     6241 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     6301 agatctgcgc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     6361 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     6421 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     6481 tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     6541 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     6601 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     6661 actcaaccct atctcggtct attcttttga tttataaggg attttgggga tttcggccta
     6721 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     6781 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     6841 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     6901 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     6961 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     7021 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     7081 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     7141 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     7201 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     7261 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     7321 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     7381 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     7441 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     7501 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     7561 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     7621 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     7681 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     7741 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     7801 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     7861 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     7921 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     7981 ggttcgcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     8041 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     8101 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     8161 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     8221 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     8281 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     8341 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     8401 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     8461 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     8521 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     8581 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     8641 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     8701 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     8761 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     8821 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     8881 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     8941 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     9001 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     9061 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     9121 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     9181 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     9241 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     9301 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     9361 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     9421 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     9481 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     9541 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     9601 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     9661 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     9721 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     9781 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     9841 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     9901 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     9961 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
    10021 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
    10081 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
    10141 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
    10201 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
    10261 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
    10321 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
    10381 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
    10441 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
    10501 tggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
    10561 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
    10621 ggcttgaccg acaattgcat gaagaatctg cttagg
//
