LOCUS       V027744                 8224 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V027744
VERSION     V027744
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             1009..3747
                     /codon_start=1
                     /product="DNA methyltransferase 3A"
                     /transl_table=1
                     /translation="MPAMPSSGPGDTSSSAAEREEDRKDGEEQEEPRGKEERQEPSTTA
                     RKVGRPGRKRKHPPVESGDTPKDPAVISKSPSMAQDSGASELLPNGDLEKRSEPQPEEG
                     SPAGGQKGGAPAEGEGAAETLPEASRAVENGCCTPKEGRGAPAEAGKEQKETNIESMKM
                     EGSRGRLRGGLGWESSLRQRPMPRLTFQAGDPYYISKRKRDEWLARWKREAEKKAKVIA
                     GMNAVEENQGPGESQKVEEASPPAVQQPTDPASPTVATTPEPVGSDAGDKNATKAGDDE
                     PEYEDGRGFGIGELVWGKLRGFSWWPGRIVSWWMTGRSRAAEGTRWVMWFGDGKFSVVC
                     VEKLMPLSSFCSAFHQATYNKQPMYRKAIYEVLQVASSRAGKLFPVCHDSDESDTAKAV
                     EVQNKPMIEWALGGFQPSGPKGLEPPEEEKNPYKEVYTDMWVEPEAAAYAPPPPAKKPR
                     KSTAEKPKVKEIIDERTRERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFVGGMCQNC
                     KNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAI
                     KEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIR
                     VLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKH
                     IQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFW
                     LFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDK
                     LELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFP
                     VHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV*"
                     /label="DNMT3A"
     polyA_signal    3824..4048
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      4094..4522
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4536..4865
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             4932..5726
                     /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    5900..6021
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(6070..6086)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6094..6110
                     /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(6118..6148)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6163..6184
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6472..7057)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7228..8088)
                     /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(8089..8193)
                     /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 ggtaccgagc tcggatccat gcccgccatg
     1021 ccctccagcg gccccgggga caccagcagc tctgctgcgg agcgggagga ggaccgaaag
     1081 gacggagagg agcaggagga gccgcgtggc aaggaggagc gccaagagcc cagcaccacg
     1141 gcacggaagg tggggcggcc tgggaggaag cgcaagcacc ccccggtgga aagcggtgac
     1201 acgccaaagg accctgcggt gatctccaag tccccatcca tggcccagga ctcaggcgcc
     1261 tcagagctat tacccaatgg ggacttggag aagcggagtg agccccagcc agaggagggg
     1321 agccctgctg gggggcagaa gggcggggcc ccagcagagg gagagggtgc agctgagacc
     1381 ctgcctgaag cctcaagagc agtggaaaat ggctgctgca cccccaagga gggccgagga
     1441 gcccctgcag aagcgggcaa agaacagaag gagaccaaca tcgaatccat gaaaatggag
     1501 ggctcccggg gccggctgcg gggtggcttg ggctgggagt ccagcctccg tcagcggccc
     1561 atgccgaggc tcaccttcca ggcgggggac ccctactaca tcagcaagcg caagcgggac
     1621 gagtggctgg cacgctggaa aagggaggct gagaagaaag ccaaggtcat tgcaggaatg
     1681 aatgctgtgg aagaaaacca ggggcccggg gagtctcaga aggtggagga ggccagccct
     1741 cctgctgtgc agcagcccac tgaccccgca tcccccactg tggctaccac gcctgagccc
     1801 gtggggtccg atgctgggga caagaatgcc accaaagcag gcgatgacga gccagagtac
     1861 gaggacggcc ggggctttgg cattggggag ctggtgtggg ggaaactgcg gggcttctcc
     1921 tggtggccag gccgcattgt gtcttggtgg atgacgggcc ggagccgagc agctgaaggc
     1981 acccgctggg tcatgtggtt cggagacggc aaattctcag tggtgtgtgt tgagaagctg
     2041 atgccgctga gctcgttttg cagtgcgttc caccaggcca cgtacaacaa gcagcccatg
     2101 taccgcaaag ccatctacga ggtcctgcag gtggccagca gccgcgcggg gaagctgttc
     2161 ccggtgtgcc acgacagcga tgagagtgac actgccaagg ccgtggaggt gcagaacaag
     2221 cccatgattg aatgggccct ggggggcttc cagccttctg gccctaaggg cctggagcca
     2281 ccagaagaag agaagaatcc ctacaaagaa gtgtacacgg acatgtgggt ggaacctgag
     2341 gcagctgcct acgcaccacc tccaccagcc aaaaagcccc ggaagagcac agcggagaag
     2401 cccaaggtca aggagattat tgatgagcgc acaagagagc ggctggtgta cgaggtgcgg
     2461 cagaagtgcc ggaacattga ggacatctgc atctcctgtg ggagcctcaa tgttaccctg
     2521 gaacaccccc tcttcgttgg aggaatgtgc caaaactgca agaactgctt tctggagtgt
     2581 gcgtaccagt acgacgacga cggctaccag tcctactgca ccatctgctg tgggggccgt
     2641 gaggtgctca tgtgcggaaa caacaactgc tgcaggtgct tttgcgtgga gtgtgtggac
     2701 ctcttggtgg ggccgggggc tgcccaggca gccattaagg aagacccctg gaactgctac
     2761 atgtgcgggc acaagggtac ctacgggctg ctgcggcggc gagaggactg gccctcccgg
     2821 ctccagatgt tcttcgctaa taaccacgac caggaatttg accctccaaa ggtttaccca
     2881 cctgtcccag ctgagaagag gaagcccatc cgggtgctgt ctctctttga tggaatcgct
     2941 acagggctcc tggtgctgaa ggacttgggc attcaggtgg accgctacat tgcctcggag
     3001 gtgtgtgagg actccatcac ggtgggcatg gtgcggcacc aggggaagat catgtacgtc
     3061 ggggacgtcc gcagcgtcac acagaagcat atccaggagt ggggcccatt cgatctggtg
     3121 attgggggca gtccctgcaa tgacctctcc atcgtcaacc ctgctcgcaa gggcctctac
     3181 gagggcactg gccggctctt ctttgagttc taccgcctcc tgcatgatgc gcggcccaag
     3241 gagggagatg atcgcccctt cttctggctc tttgagaatg tggtggccat gggcgttagt
     3301 gacaagaggg acatctcgcg atttctcgag tccaaccctg tgatgattga tgccaaagaa
     3361 gtgtcagctg cacacagggc ccgctacttc tggggtaacc ttcccggtat gaacaggccg
     3421 ttggcatcca ctgtgaatga taagctggag ctgcaggagt gtctggagca tggcaggata
     3481 gccaagttca gcaaagtgag gaccattact acgaggtcaa actccataaa gcagggcaaa
     3541 gaccagcatt ttcctgtctt catgaatgag aaagaggaca tcttatggtg cactgaaatg
     3601 gaaagggtat ttggtttccc agtccactat actgacgtct ccaacatgag ccgcttggcg
     3661 aggcagagac tgctgggccg gtcatggagc gtgccagtca tccgccacct cttcgctccg
     3721 ctgaaggagt attttgcgtg tgtgtaagaa ttctgcagat atccagcaca gtggcggccg
     3781 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3841 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3901 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3961 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     4021 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     4081 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4141 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4201 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4261 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4321 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4381 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4441 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4501 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     4561 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4621 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4681 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     4741 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4801 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4861 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4921 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4981 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     5041 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     5101 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     5161 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     5221 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     5281 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5341 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5401 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     5461 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     5521 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     5581 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5641 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5701 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     5761 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5821 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5881 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5941 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     6001 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     6061 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     6121 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     6181 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6241 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     6301 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     6361 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     6421 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6481 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6541 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6601 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6661 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6721 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6781 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6841 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6901 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6961 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg
     7021 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     7081 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7141 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7201 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7261 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7321 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7381 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7441 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7501 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7561 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7621 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7681 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7741 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7801 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7861 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7921 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7981 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     8041 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     8101 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     8161 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     8221 cgtc
//
