LOCUS       V037104                 9321 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V037104
VERSION     V037104
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             991..4131
                     /label="RASA1(NM_002890)"
                     /note="RASA1(NM_002890)"
                     /gene="RASA1"
     CDS             4171..4890
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /transl_table=1
                     /translation="M,V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL
                     TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG
                     IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV
                     LLEFVTAAGITLGMDELYK*"
                     /label="EGFP"
     polyA_signal    4921..5145
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      5191..5619
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        5633..5962
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             6029..6823
                     /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    6997..7118
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(7167..7183)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    7191..7207
                     /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(7215..7245)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    7260..7281
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(7569..8154)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(8325..9185)
                     /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(9186..9290)
                     /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 atgatggcgg ccgaggccgg cagtgaggag
     1021 ggcggcccgg taacagccgg agctggagga ggcggcgcgg cagcgggctc cagtgcctat
     1081 cccgcagtgt gtcgggtgaa gatacccgcg gccctgcctg tggcagccgc cccctatcct
     1141 gggctggtgg agaccggagt ggctggaact ctgggtggcg gagccgcttt ggggtcagag
     1201 ttcctaggag ccgggtctgt ggcaggggca ctggggggag ctggactgac agggggaggt
     1261 actgctgctg gcgtagctgg tgctgctgct ggcgtggccg gtgctgctgt tgctggacct
     1321 agtggagaca tggctctcac caaactgccc acttcgttgc ttgctgagac tctcgggcca
     1381 ggcggcggtt ttccccctct gccccctccc ccttacctgc cccctttggg ggcgggcctc
     1441 gggacagtgg acgaaggtga ctctctggat ggaccagaat acgaggagga agaggtggcc
     1501 ataccgttga ccgctcctcc aactaaccag tggtatcacg gaaaacttga cagaacgata
     1561 gcagaagaac gcctcaggca ggcagggaag tctggcagtt atcttataag agagagtgat
     1621 cggaggccag ggtcctttgt actttcattt cttagccaga tgaatgttgt caaccatttt
     1681 aggattattg ctatgtgtgg agattactac attggtggaa gacgtttttc ttcactgtca
     1741 gacctaatag gttattacag tcatgtttct tgtttgctta aaggagaaaa attactttac
     1801 ccagttgcac caccagagcc agtagaagat agaaggcgtg tacgagctat tctaccttac
     1861 acaaaagtac cagacactga tgaaataagt ttcttaaaag gagatatgtt cattgttcat
     1921 aatgaattag aagatggatg gatgtgggtt acaaatttaa gaacagatga acaaggcctt
     1981 attgttgaag acctagtaga agaggtgggc cgggaagaag atccacatga aggaaaaata
     2041 tggttccatg ggaagatttc caaacaggaa gcttataatt tactaatgac agttggtcaa
     2101 gtctgcagtt ttcttgtgag gccctcagat aatactcctg gcgattattc actttatttc
     2161 cggaccaatg aaaatattca gcgatttaaa atatgtccaa cgccaaacaa tcagtttatg
     2221 atgggaggcc ggtattataa cagcattggg gacatcatag atcactatcg aaaagaacag
     2281 attgttgaag gatattatct taaggaacct gtaccaatgc aggatcaaga acaagtactc
     2341 aatgacacag tggatggcaa ggaaatctat aataccatcc gtcgtaaaac aaaggatgcc
     2401 ttttataaaa acattgttaa gaaaggttat cttctgaaaa agggcaaagg aaaacgttgg
     2461 aaaaatttat attttatctt agagggtagt gatgcccaac ttatttattt tgaaagcgaa
     2521 aaacgagcta ccaaaccaaa aggattaata gatctcagtg tatgttctgt ctatgtcgtt
     2581 catgatagtc tctttggcag gccaaactgt tttcagatag tagttcagca ctttagtgaa
     2641 gaacattaca tcttttactt tgcaggagaa actccagaac aagcagagga ttggatgaaa
     2701 ggtctgcagg cattttgcaa tttacggaaa agtagtccag ggacatccaa taaacgcctt
     2761 cgtcaggtca gcagccttgt tttacatatt gaagaagccc ataaactccc agtaaaacat
     2821 tttactaatc catattgtaa catctacctg aatagtgtcc aagtagcaaa aactcatgca
     2881 agggaagggc aaaacccagt atggtcagaa gagtttgtct ttgatgatct tcctcctgac
     2941 atcaatagat ttgaaataac tcttagtaat aaaacaaaga aaagcaaaga tcctgatatc
     3001 ttatttatgc gctgccagtt gagccgatta cagaaagggc atgccacaga tgaatggttt
     3061 ctgctcagct cccatatacc attaaaaggt attgaaccag ggtccctgcg tgttcgagca
     3121 cgatactcta tggaaaaaat catgccagaa gaagagtaca gtgaatttaa agagcttata
     3181 ctgcaaaagg aacttcatgt agtctatgct ttatcacatg tatgtggaca agaccgaaca
     3241 ctactggcca gcatcctact gaggattttt cttcacgaaa agcttgaatc gttgttgtta
     3301 tgcacactaa atgacagaga aataagcatg gaagatgaag ccactaccct atttcgagcc
     3361 acaacacttg caagcacctt gatggagcag tatatgaaag ccactgctac acagtttgtt
     3421 catcatgctt tgaaagactc tattttaaag ataatggaaa gcaagcagtc ttgtgagtta
     3481 agtccatcaa agttagaaaa aaatgaagat gtgaacacta atttaacaca cctattgaac
     3541 atactttcag agcttgtgga gaaaatattc atggcttcag aaatacttcc accgacattg
     3601 agatatattt atgggtgttt acagaaatct gttcagcata agtggcctac aaataccacc
     3661 atgagaacaa gagttgttag tggttttgtt tttcttcgac tcatctgtcc tgccatcctg
     3721 aatccacgga tgttcaatat catctcagat tctccatctc ctattgctgc aagaacactg
     3781 atattagtgg ctaaatctgt gcagaactta gcaaatcttg tggaatttgg agctaaggag
     3841 ccctacatgg aaggtgtcaa tccattcatc aaaagcaaca aacatcgtat gatcatgttt
     3901 ttagatgaac ttgggaatgt acctgaactt ccggacacta cagagcattc tagaacggac
     3961 ctgtcccgtg atttagcagc attgcatgag atttgcgtgg ctcattcaga tgaacttcga
     4021 acgctcagta atgagcgtgg tgcacagcag cacgtattga aaaagcttct ggctataaca
     4081 gaactgcttc aacaaaaaca aaaccagtat acaaaaacca atgatgtcag gacctcgagt
     4141 ggcggtggag gaagcggcgg aggtggatct atggtgagca agggcgagga gctgttcacc
     4201 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     4261 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     4321 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     4381 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     4441 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     4501 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     4561 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     4621 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     4681 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     4741 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     4801 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     4861 actctcggca tggacgagct gtacaagtag ggcccgttta aacccgctga tcagcctcga
     4921 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     4981 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5041 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5101 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     5161 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     5221 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5281 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5341 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5401 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5461 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5521 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5581 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     5641 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     5701 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     5761 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     5821 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     5881 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     5941 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     6001 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     6061 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     6121 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     6181 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     6241 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     6301 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     6361 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     6421 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     6481 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     6541 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     6601 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     6661 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     6721 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     6781 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     6841 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     6901 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     6961 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     7021 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     7081 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     7141 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     7201 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     7261 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     7321 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     7381 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     7441 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     7501 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     7561 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     7621 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     7681 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     7741 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     7801 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     7861 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     7921 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     7981 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     8041 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     8101 agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     8161 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     8221 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     8281 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     8341 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     8401 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     8461 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     8521 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     8581 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     8641 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     8701 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     8761 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     8821 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     8881 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     8941 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     9001 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     9061 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     9121 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     9181 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     9241 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     9301 cgaaaagtgc cacctgacgt c
//
