LOCUS       V032168                 8671 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032168
VERSION     V032168
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"
     CDS             964..4161
                     /label="Rnf31(NM_194346)"
                     /note="Rnf31(NM_194346)"
                     /gene="Rnf31"
     CDS             4168..4233
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     polyA_signal    4271..4495
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      4541..4969
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4983..5312
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             5379..6173
                     /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    6347..6468
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(6517..6533)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6541..6557
                     /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(6565..6595)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6610..6631
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6919..7504)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7675..8535)
                     /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(8536..8640)
                     /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 aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcgcc
      961 accatgccgg gagacgagga gcgaggcttc ctggcggccc gcgaggagct ggcgagcgcc
     1021 ctgaggtggg attctgcgca ggtttttccc ctggagcagc tcatgccgct tctggccacc
     1081 tctctgccac cagccgcccg ctacctgcag ctggacgccg gacgcttggt ccgctgcaac
     1141 gctcatgggg agcctcgaaa ctacctcaac accctatcca cggccctgaa catcctggaa
     1201 aaatatggtc gcaacctcct cagcccgcag cggccccggt attggcgctc agtgaagttt
     1261 aataaccccg tctttcgcag cacggtggat gctgtgcagg gtggccggga tgtactacgg
     1321 ttgtatggct atactgagga gcgcccagat ggattgagtt tccccgaagg gcaggaggaa
     1381 ccagatgaat accaggttgc tgttgtcaca ctagaagtac tgctgcttcg caccgagctc
     1441 agtttgctgt tgcagaatac tcatcccaga cagaatgcac tggaccagct gctaagagag
     1501 agcgttgaag atggtatgct gcagctttca gagtttcacc cccttctgag ggagattgtt
     1561 cctggccccc gcccctctgc ccaaggctcc actcctggtc cctgtttcct ctgtggttct
     1621 gccccaggca cactgcactg tccagcctgt aaccaagtct cgtgcccagc ttgtgacatt
     1681 ttgttccatg ggcatccgtc ccgtgcacat caccttcgcc aagccctgcc tgggtcccac
     1741 cagactgcca gcctgagctc tagtttacct gcctcgtccc aaccacggcc cccctcctcc
     1801 tccttggccc tgggagatag ctctctttct tcccctgacc ctgcaaatgc ctgtctgccc
     1861 tggcattgtc ttacctgtgc cacactaaat gagccttggg cagtgttctg tgcagtctgt
     1921 agtcagccca aaggctgcaa ggtgccggga atagagggtt cccatggaac cgggggccta
     1981 gaacctgagc ctgcacggga tcaatgggcc tgccagagct gtacctttga gaatgaggca
     2041 gcagctgtgc tatgcgccat atgtgagcga cctcggctgg cccagcctcc cagcttggtg
     2101 gtggattccc atgatgctgg tgtttgccaa cagtccctta agcaggagga tcctttgctc
     2161 accgctgccc agcctcaggt gtggtactgt gaccattgta ccttctgcaa ttcaggccct
     2221 gtctgggtgt gtgccatgtg caaccgaacc cgagacccca tccctacaca gcctgccctc
     2281 cagtcctatc ccagctcttt ggaaaaggga cgcccaaagc cagggtcctc acaacacctt
     2341 ggttcctccc tgcctgcttc ctgtggagac ccagagaaac aacgccaaga taagatgcgg
     2401 aaggaaggtc tccagctcgt gagcatgatc caggaaggag aaactgcggg tgccagtcca
     2461 gaagaggtct tctcagctct ccaatactca ggcacagagg tgcccctcca gtggttgcgt
     2521 tcagagctgt cctacgtcct ggagatggtg gctgagcttg ctggacaaca ggatccagag
     2581 ctgggggcct tttcctgtca ggaagcccgg aaagcctggc ttgatcgcca tggcaacctg
     2641 gatgaagctg tagaggagtg tgtgagggcc aggaggagga aggtgcacga gctgcagtcc
     2701 ctgggctttg ggcctaagga agggtcacta caggcattgt tccagcatgg gggtgacgtg
     2761 gctcgggccc tgactgagtt acagcgccag cgcctggagc ccttccatca gcgcctatgg
     2821 gacagagacc ctgaacccac tccctgctgg gatgggctgg acagacagag cttggtcaga
     2881 cgccttctgg ccgtctacac actccccagc tggggccgag cagagctggc gctggcgctg
     2941 ctgcaggaga cacccaggaa ctatgagttg ttggacgtgg tggaggctgt gaggcacagc
     3001 caggaccggg cctttctgcg tcgactgctt gcccaggaat gtgctgtgtg cgggtgggcc
     3061 cttccccgca accggatgca ggccctgatc tcctgtgagt gcaccatatg tcccgaatgc
     3121 ttccgccaac acttcaccat tgccctgaag gagaagcaca tcacagacat ggtgtgccct
     3181 gcctgtggcc gccctgacct cactgatgac gctcagttac tcagctactt ctccaccctt
     3241 gacatccagc tcagagagag cctagacccc gatgcatatg ccctgtttca caagaagctg
     3301 accgaggctg tgcttatgcg agaccccaag ttcttgtggt gcgcccagtg ttcctttggc
     3361 ttcatctatg aacgcgaaca gctggaggcg acgtgtcccc agtgtcacca gaccttctgt
     3421 gtgcgctgca agcgccagtg ggaggagcag cacagaggac ggagttgtga ggatttccag
     3481 aactggaaac gcaccaatga cccagagtac caggctcaag gcttggccat gtaccttcag
     3541 gaaaacggca ttgactgtcc gaaatgcaag ttctcgtacg cactggcccg gggaggctgc
     3601 atgcacttcc actgcacgca gtgtcgacac cagttctgca gtggctgcta caacgccttt
     3661 tacgccaaga ataaatgtcc agaccctaac tgcaaggtga aaaagtccct gcatggccac
     3721 caccctcgag actgcctctt ctacctacgg gactggactg ctgcccgcct ccagaaactg
     3781 ttgcaggaca ataatgtcat gtttaataca gagcctccag ctgggacacg ggcagtccct
     3841 ggagggggct gcagagtgat ggagcagaag gaggtccata gtgggttcag ggatgaagct
     3901 tgcggcaagg aaactccacc tggctatgcc ggcctatgtc aggcacacta caaagagtat
     3961 ctcgtgagcc tcatcaatgc ccattcactg gacccagcta ccttgtatga agtggaggag
     4021 ctggagacag ccactattcg ctacctacat ttagctcctc agcccgcgga tggagaggat
     4081 ctgcctgctt accaggcccg gctattacag aagctgagag aagaggtacc cttgggacag
     4141 agtattgccc gcagaagaaa gctcgaggac tacaaagacc atgacggtga ttataaagat
     4201 catgacatcg actacaagga tgacgatgac aagtagtgag ggcccgttta aacccgctga
     4261 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     4321 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     4381 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     4441 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     4501 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     4561 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4621 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4681 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4741 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4801 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4861 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4921 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     4981 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     5041 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     5101 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     5161 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5221 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     5281 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     5341 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     5401 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     5461 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     5521 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     5581 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     5641 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     5701 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     5761 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     5821 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     5881 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     5941 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     6001 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     6061 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     6121 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     6181 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     6241 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     6301 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     6361 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     6421 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     6481 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     6541 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     6601 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     6661 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6721 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     6781 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6841 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6901 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6961 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     7021 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     7081 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     7141 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     7201 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     7261 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     7321 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     7381 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     7441 caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7501 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     7561 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7621 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     7681 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7741 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     7801 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     7861 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     7921 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     7981 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     8041 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     8101 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     8161 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     8221 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     8281 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     8341 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     8401 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     8461 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     8521 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     8581 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     8641 cacatttccc cgaaaagtgc cacctgacgt c
//
