LOCUS       V032650                 9807 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032650
VERSION     V032650
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        202..581
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        582..785
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        830..848
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             872..4576
                     /label="Irs1(NM_012969)"
                     /note="Irs1(NM_012969)"
                     /gene="Irs1"
     CDS             4613..5323
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /transl_table=1
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK*"
                     /label="mCherry"
     polyA_signal    5374..5598
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      5644..6072
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        6086..6415
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             6482..7276
                     /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    7450..7571
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(7620..7636)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    7644..7660
                     /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(7668..7698)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    7713..7734
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(8022..8607)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(8778..9638)
                     /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(9639..9743)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
       61 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      121 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      181 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      241 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      301 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      361 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      421 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      481 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      541 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      601 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      661 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      721 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      781 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
      841 actataggga gacccaagct ggctagccac catggcgagc cctccggata ccgatggctt
      901 ctcagacgtg cgcaaggtgg gttacctgcg caaacccaag agtatgcata agcgcttttt
      961 cgtgctgcgg gcggccagcg aggccggggg cccggcgcgc ctggagtatt atgagaacga
     1021 gaagaagtgg cggcacaagt cgagcgcccc caaacgctcg atccccctcg agagctgttt
     1081 caacatcaac aagcgggctg actccaagaa caagcacctg gtggctctct acacccgaga
     1141 cgaacacttt gccattgcgg cggatagcga ggctgaacaa gacagctggt accaggctct
     1201 tctgcagctg cataatcggg caaaggccca ccatgacggg gctggaggag gctgcggtgg
     1261 tagctgcagc ggcagctctg gcgtcggaga ggcaggggag gacttgagct atgacacggg
     1321 cccaggaccc gcgttcaagg aggtctggca ggttatcctg aaacccaagg gcttaggtca
     1381 gacaaagaac ttgattggta tctaccgcct ctgcctgacc agcaagacca tcagctttgt
     1441 gaagctcaac tctgaggctg ccgctgtggt gctgcagctg atgaacatca gacgctgtgg
     1501 ccactcagag aacttcttct tcatcgaggt ggggcggtca gctgtgaccg ggcccggcga
     1561 gttctggatg caagtggatg actccgtggt ggcccagaac atgcatgaga ccattctaga
     1621 ggccatgcgg gccatgagcg atgagtttcg cccgcgcagc aaaagccaat cttcatccag
     1681 ttgctccaac cccatcagtg ttcccctgcg caggcaccat ctcaacaatc ctccgcccag
     1741 ccaagtgggg ctgactcgga gatctcgaac tgagagcatc actgccacct cccctgccag
     1801 tatggtgggt gggaaaccag gttccttcag ggtgcgtgcc tccagcgatg gcgaaggcac
     1861 catgtcccgt ccagcatcag tggatggcag tcctgtgagc cctagcacca acaggaccca
     1921 cgcccatcgg catcgaggca gctccaggtt gcacccccca ctcaaccaca gccgctccat
     1981 ccctatgcct tcttcacgat gctccccttc agccaccagc ccagtgagcc tgtcatccag
     2041 tagtaccagt ggccacggct ccacttcaga ctgtctcttc ccgaggcgct ctagtgcttc
     2101 cgtgtccggt tctcctagcg atggcggttt catctcttct gatgagtatg gctctagtcc
     2161 ctgcgatttc cgaagttcct tccgcagtgt caccccagat tccctgggcc acaccccacc
     2221 agccaggggt gaggaggagc tgagcaacta tatctgcatg ggtggcaagg gagcctccac
     2281 cttgacagct cccaatggtc actacatttt gtctaggggt ggcaacggcc atcgctacat
     2341 cccaggtgct accatgggga caagcccggc gctgactgga gacgaagccg ctggtgcagc
     2401 agatctggat aaccggtttc ggaagagaac tcactcggct ggcacgtccc ccaccatatc
     2461 ccaccagaag accccctcgc agtcctcagt ggtttctatt gaggaatata cagagatgat
     2521 gcccgctgcc tacccaccag gaggtggcag tggaggccga ctgcccggct accggcattc
     2581 cgccttcgtg cccacccact cctatcccga ggagggtcta gagatgcacc acttggaacg
     2641 tcgtgggggc caccaccgtc cagactcctc caacctccac accgatgatg gctacatgcc
     2701 catgtctccc ggagtggctc cagtgcccag caaccgcaaa ggaaatgggg actatatgcc
     2761 catgagcccc aagagtgtat ctgcccccca gcagatcatt aaccccatca ggcgccaccc
     2821 acagagagtg gaccccaatg gctacatgat gatgtctccc agtggtagtt gctctcctga
     2881 cattggaggt gggtcttgca gcagtagcag catcagcgca gccccttctg ggagcagcta
     2941 tgggaagcca tggacaaacg gagtaggggg gcaccatacc catgcccttc cccatgccaa
     3001 acctcctgtt gagagcggtg gtggtaagct cttgccttgc actggtgact acatgaacat
     3061 gtcgccagtg ggagattcca acaccagcag cccctcagaa tgctactatg gcccagaaga
     3121 tccccagcac aagcctgtcc tctcctacta ctcattacca aggtccttta agcacaccca
     3181 gcgccctggg gagccagagg agggtgccag gcaccagcat cttcgtctct cttcaagctc
     3241 tggacgcctt cgctataccg caactgccga agattcctcc tcttccacca gcagcgacag
     3301 cctgggtggg ggttactgtg gggctaggcc agagtctagc gtcacacatc cccaccacca
     3361 tgccttgcag ccccatctgc ctcgaaaggt agacacagct gcacagacca acagccgcct
     3421 ggctcgaccc acaaggctgt ccttggggga tcccaaggca agcactttac cccgggtacg
     3481 agagcaacag cagcagcagc aacagcagca gcagtcttcc ctgcaccctc ccgagcccaa
     3541 aagcccagga gaatatgtga atattgaatt cgggagtggc cagccaggct atttagctgg
     3601 ccctgcaact tcccgtagct ccccttcagt tcgatgtcta ccccagctcc acccagctcc
     3661 cagagaagag actggctcgg aagagtacat gaacatggac ttggggccag gccggagggc
     3721 aacctggcag gagagtggtg gagttgagtt gggcagagta ggccctgcac ctccaggggc
     3781 tgcttccatt tgtaggccaa cccggtcggt gccaaatagc cgtggtgatt acatgaccat
     3841 gcagataggt tgtcctcgtc aaagctatgt ggatacctca ccagtggccc cagtcagcta
     3901 tgctgacatg cggacaggca ttgctgcaga gaaggtgagc ctgcccagaa ccacaggagc
     3961 tgccccccct ccatcctcca cagcctctgc ttctgcttct gttacacctc aaggggccgc
     4021 tgagcaggcc gctcactctt ccttgctggg aggccctcag ggacctgggg gcatgagcgc
     4081 attcaccagg gtgaacctaa gtcccaacca taaccagagt gccaaagtga ttcgtgcaga
     4141 cactcaaggc tgccggagga ggcacagctc cgagaccttc tcggcgccta cgcgggctgg
     4201 caacacagtg tcttttggag caggggctgc aggagggggc agcggtggtg gcagtgagga
     4261 tgtgaaacgc cacagctctg catcctttga gaatgtgtgg ctgagacccg gggatctagg
     4321 gggagcatcc aaggagtcgg ctccagggta cggggctgcc gggggattgg agaagagtct
     4381 taactatata gacttggatt tggtcaagga tgttaagcag caccctcaag actgcccctc
     4441 tcaacagcag tccctgccac cccctccccc tcaccaaccc ttaggcagca atgagggcag
     4501 ctccccaaga cgctccagtg aggatttaag cacctatgcc agcatcaact tccagaagca
     4561 accagaggac cgtcaagaat tcggtggagg cggatcagga ggcggtggat ctatggtgag
     4621 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     4681 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     4741 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     4801 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     4861 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     4921 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     4981 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     5041 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     5101 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     5161 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     5221 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     5281 gggccgccac tccaccggcg gcatggacga gctgtacaag taggcggccg ctcgagtcta
     5341 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     5401 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     5461 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     5521 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     5581 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     5641 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     5701 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     5761 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     5821 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     5881 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5941 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     6001 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     6061 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     6121 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     6181 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     6241 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     6301 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     6361 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     6421 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     6481 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     6541 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     6601 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     6661 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     6721 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     6781 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     6841 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     6901 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     6961 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     7021 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     7081 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     7141 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     7201 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     7261 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     7321 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     7381 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     7441 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     7501 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     7561 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     7621 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     7681 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     7741 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     7801 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     7861 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     7921 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     7981 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     8041 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     8101 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     8161 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     8221 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     8281 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     8341 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     8401 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     8461 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     8521 agctcttgat ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg caagcagcag
     8581 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     8641 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     8701 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     8761 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     8821 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     8881 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     8941 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     9001 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     9061 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     9121 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     9181 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     9241 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     9301 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     9361 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     9421 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     9481 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     9541 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     9601 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     9661 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     9721 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     9781 tcgggagatc tcccgatccc ctatggt
//
