LOCUS       Exported               10788 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  noninfectious (non-packaging), self-replicating Venezuelan equine 
            encephalitis (VEE) virus RNA replicon.
ACCESSION   .
VERSION     .
KEYWORDS    SP6-VEE-IRES-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10788)
  AUTHORS   Yoshioka N, Gros E, Li HR, Kumar S, Deacon DC, Maron C, Muotri AR, 
            Chi NC, Fu XD, Yu BD, Dowdy SF
  TITLE     Efficient generation of human iPSCs by a synthetic self-replicative 
            RNA.
  JOURNAL   Cell Stem Cell. 2013 Aug 1;13(2):246-54. doi: 
            10.1016/j.stem.2013.06.001.
  PUBMED    23910086
REFERENCE   2  (bases 1 to 10788)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10788
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     RBS             1535..1543
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(7820..7837)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     8047..8066
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             8228..8827
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(8228..8247)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8724..8744
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     promoter        9025..9129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             9130..9990
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9348..9367)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10161..10749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10650..10669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        10771..1
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     10771..10788
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 atgggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg
       61 ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg
      121 aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc
      181 tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa
      241 gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat
      301 gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg
      361 aaataactga taaggaattg gacaagaaaa tgaaggagct ggccgccgtc atgagcgacc
      421 ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc
      481 aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag
      541 ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta
      601 agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa
      661 cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt
      721 ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga
      781 ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact
      841 tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg
      901 tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta
      961 cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg
     1021 tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac
     1081 tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta
     1141 tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg
     1201 tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa
     1261 ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc
     1321 acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg
     1381 atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa
     1441 caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg
     1501 acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt
     1561 tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg
     1621 tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa
     1681 aggttaccag ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg
     1741 ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga
     1801 taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg
     1861 tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca
     1921 ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag
     1981 gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg
     2041 aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag
     2101 ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa
     2161 cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag
     2221 gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga
     2281 aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg
     2341 ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata
     2401 ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac
     2461 ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc
     2521 tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc
     2581 gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa
     2641 cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc
     2701 aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca
     2761 aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg
     2821 ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg
     2881 tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga
     2941 taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag
     3001 cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc
     3061 agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca
     3121 tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact
     3181 cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg
     3241 gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc
     3301 cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc
     3361 cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc
     3421 gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag
     3481 tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg
     3541 gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt
     3601 tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg
     3661 tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc
     3721 agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc
     3781 tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa
     3841 gcatcattgg tgctatagcg cggcagttca agttttcccg ggtatgcaaa ccgaaatcct
     3901 cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc
     3961 acaattctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg
     4021 aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag
     4081 gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc
     4141 tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac
     4201 tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt
     4261 cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca
     4321 acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga
     4381 acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg
     4441 cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg
     4501 ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg
     4561 atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca
     4621 caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg
     4681 atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca
     4741 tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg
     4801 aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa
     4861 gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat
     4921 tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct
     4981 caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag
     5041 acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac
     5101 cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg
     5161 aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg
     5221 aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat
     5281 ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca
     5341 gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc
     5401 gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa
     5461 gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc
     5521 caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc
     5581 ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga
     5641 ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg
     5701 catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa
     5761 cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc
     5821 tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta
     5881 acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta
     5941 ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc
     6001 tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg
     6061 cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta
     6121 ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca
     6181 ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac
     6241 ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag
     6301 ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg
     6361 cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt
     6421 ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa
     6481 aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca
     6541 taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa
     6601 aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag
     6661 cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga
     6721 acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact
     6781 tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg
     6841 acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt
     6901 tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta
     6961 aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag
     7021 tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg
     7081 cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag
     7141 acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga
     7201 aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc
     7261 gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg
     7321 aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg
     7381 gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca
     7441 tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag
     7501 gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa
     7561 gtctagcata tgggcgcgcc ctcagcatcg attcaattgg gccggccgcg gccgcaacta
     7621 acttaagcta gcaacggttt ccctctagcg ggatcaattc cgcccccccc ccctaacgtt
     7681 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     7741 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     7801 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     7861 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     7921 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     7981 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     8041 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     8101 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     8161 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     8221 taataccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     8281 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     8341 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     8401 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     8461 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     8521 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     8581 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     8641 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     8701 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     8761 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     8821 tgcctgagaa ttggcaagct gcttacatag aactcgcggc gattggcatg ccgccttaaa
     8881 atttttattt tatttttctt ttcttttccg aatcggattt tgtttttaat atttcaaaaa
     8941 aaaaaaaaaa aaaaaaaaaa acgcgtcgag gggaattaat tcttgaagac gaaagggcca
     9001 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     9061 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     9121 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     9181 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     9241 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     9301 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     9361 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     9421 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     9481 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     9541 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     9601 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     9661 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     9721 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     9781 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     9841 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     9901 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     9961 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
    10021 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
    10081 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
    10141 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
    10201 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
    10261 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
    10321 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
    10381 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
    10441 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
    10501 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa
    10561 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
    10621 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
    10681 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
    10741 ctatggaaaa acgccagcaa cgcgagctcg atttaggtga cactatag
//
