LOCUS       V031330                 7132 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V031330
VERSION     V031330
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             997..2652
                     /label="Smad4(NM_019275)"
                     /note="Smad4(NM_019275)"
                     /gene="Smad4"
     polyA_signal    2732..2956
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      3002..3430
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        3444..3773
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             3840..4634
                     /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    4808..4929
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(4978..4994)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5002..5018
                     /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(5026..5056)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5071..5092
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(5380..5965)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6136..6996)
                     /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(6997..7101)
                     /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 ggtaccatgg acaatatgtc tattacaaat
     1021 acaccgacaa gcaatgacgc ctgtctgagc attgtacaca gtttgatgtg ccacagacaa
     1081 ggtggagaga gtgagacatt tgccaaaaga gcaattgaga gtctggtaaa gaagctgaaa
     1141 gagaagaaag atgaactgga ttctttaata acagctatca ctacaaatgg agctcacccc
     1201 agcaagtgtg tcaccataca gagaacactg gatggacgac tgcaggtggc tggtcggaaa
     1261 ggatttcctc atgtgatcta tgcccgtctg tggaggtggc ctgatctaca caagaacgaa
     1321 ctaaaacatg ttaaatattg tcagtatgca tttgacttaa aatgtgatag cgtctgtgtg
     1381 aacccatatc actatgagcg ggttgtctca cctggaattg atctctctgg attaacactg
     1441 cagagtaatg ctccaccaag tatgttagtg aaggatgaat atgttcatga ctttgaagga
     1501 cagccatcct tacccacgga aggacattcg attcaaacca tccaacatcc accaagtaat
     1561 cgcgcatcca cggagacgta cagcgcccca gctctgttag ccccatcaga gtctaatgcc
     1621 accagtacca ccaacttccc caacattcct gtggcttcca caagtcagcc ggccagcatt
     1681 ctggcaggca gccatagtga aggactgttg cagatagctt cagggcctca gccaggacag
     1741 cagcagaatg gattcactgc tcagccagct acttaccacc ataacagcac taccacctgg
     1801 actggaagta ggactgcacc atacacacct aatttgcctc accaccaaaa cggccatctt
     1861 cagcaccacc cgcctatgcc gccccatcct ggacattact ggccggttca caatgagctt
     1921 gcattccagc ctcccatttc caatcatcct gctcctgaat attggtgttc cattgcttac
     1981 tttgaaatgg atgttcaggt aggagagaca tttaaagtcc cttcaagctg tcctattgta
     2041 actgtggatg gatacgtgga tccttctgga ggagatcgct tttgcttggg tcaactctcc
     2101 aatgtccaca ggacagaagc tattgagaga gcaaggttgc acataggcaa aggagtgcag
     2161 ttggagtgta aaggtgaagg ggacgtgtgg gtcaggtgcc tcagtgacca cgccgtcttc
     2221 gtgcagagtt actacctgga ccgtgaagct ggccgagcac ctggtgacgc cgtccacaag
     2281 atctacccga gtgcgtatat aaaggtcttt gatttgcggc agtgtcaccg gcagatgcag
     2341 cagcaggcgg ccaccgcgca agccgcagct gctgcccagg cggcagctgt ggcaggaaac
     2401 atccctggtc ctgggtccgt aggtggaata gccccggcca tcagtctgtc tgctgctgct
     2461 ggcattggtg tagacgacct ccggcgcttg tgcatactca ggatgagctt cgtgaagggc
     2521 tggggcccag actacccgag acagagcatc aaagaaaccc cgtgctggat tgagattcac
     2581 ctccaccgag ctctgcagct cctggatgaa gtcctgcaca ccatgcccat tgcggaccca
     2641 cagcctttag actgagaatt ctgcagatat ccagcacagt ggcggccgct cgagtctaga
     2701 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2761 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2821 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2881 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2941 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     3001 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3061 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3301 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3361 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3421 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3481 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3541 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3601 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3661 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3721 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3781 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     3841 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3901 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3961 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4021 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4081 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4141 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4201 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4261 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4321 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4381 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4441 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4501 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4561 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4621 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4681 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4741 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4801 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4861 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4921 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     4981 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5041 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5101 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5161 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5221 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5281 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5341 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5401 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5461 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5521 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5581 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5641 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5701 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5761 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5821 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5881 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca agcagcagat
     5941 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6001 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6061 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6121 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6181 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6241 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6301 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6361 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6421 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6481 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6541 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6601 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6661 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6721 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6781 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6841 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6901 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6961 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7021 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7081 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
