LOCUS       sequence-410193-        6576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enables expression of an ORF (with or without tags) and selection 
            with puromycin in insect cells. Includes GFP-T2A-puro with Actin5C 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6576)
  AUTHORS   Gonzalez M, Martin-Ruiz I, Jimenez S, Pirone L, Barrio R, Sutherland
            JD
  TITLE     Generation of stable Drosophila cell lines using multicistronic 
            vectors.
  JOURNAL   Sci Rep. 2011;1:75. doi: 10.1038/srep00075. Epub 2011 Aug 31.
  PUBMED    22355594
REFERENCE   2  (bases 1 to 6576)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2011"; volume: "1:75. doi"; pages: " 10.1038/srep00075. Epub 
            2011 Aug 31"
COMMENT     Sequence Label: Ac5-STABLE1-puro
FEATURES             Location/Qualifiers
     source          1..6576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      2481..2490
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2487..2489,2490..2492,2493..3203)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKGD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2532..2553)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3140..3161
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3207..3260
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3285..3884
                     /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(3285..3304)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3781..3801
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3892..4026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3916..3935)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3970..3989
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(4627..5215)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4707..4726)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5386..6177,6178..6246))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6009..6028
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6247..6351)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6419..6437
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(6501..6576,1..2434)
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
ORIGIN
        1 agatctcgct gcctgttatg tggcccacaa accaagacac gttttatggc cattaaagct
       61 ggctgatcgt cgccaaacac caaatacata atgaatatgt acacattcga gaaagaagcg
      121 atcaaagaag cgtcttcggg cggagtagga gaatgcggag gagaaggaga acgagctgat
      181 ctagtatctc tccacaatcc aatgccaact gaccaactgg ccatattcgg agcaatttga
      241 agccaatttc catcgcctgg cgatcgctcc attcttggct atatgttttt caccgttacc
      301 cggggccatt ttcaaagact cgtcggcaag ataagattgt gtcactcgct gtctctcttc
      361 atttgtcgaa gaatgctgag gaatttcgcg atgacgtcgg cgagtatttt gaagaatgag
      421 aataatttgt atttatacga aaatcagtta gtggaatttt ctacaaaaac atgttatcta
      481 tagataattt tgttgcaaaa tatgttgact atgacaaaga ttgtatgtat atacctttaa
      541 tgtattctca ttttcttatg tatttataat ggcaatgatg atactgatga tattttaaga
      601 tgatgccaga ccaaaaggct tgaatttctg cgtcttttgc cgaacgcagt gcatgtgcaa
      661 ttgttgtttt ttggaatatt caattttcgg actgtccgct ttgatttcag tttcttggct
      721 tattcaaaaa gcaaagtaaa gccaaaaaag cgagatggca ataccaaatg cggcaaaacg
      781 gtagtggaag gaaaggggtg cggggcagcg gaaggaaggg tggggcgggg cgtggcgggg
      841 tctgtggctg ggcgcgacgt caccgacgtt ggagccactc ctttgaccat gtgtgcgtgt
      901 gtgtattatt cgtgtctcgc cactcgccgg ttgttttttt ctttttatgc tgcgctctct
      961 ctagcgccat ctcgcttacg catgctcaac gcaccgcatg ttgccgtttc cttttatgcg
     1021 tcattttggc tcgaaatagg caattattta aacaaagatt agtcaacgaa aacgctaaaa
     1081 taaataagtc tacaatatgg ttacttattg ccatgtgtgt gcagccaacg atagcaacaa
     1141 aagcaacaac acaggtggct ttccctcttt cactttttgt ttgcaagccg cgtgcgagca
     1201 agacggcacg accggcaaac gcaattacgc tgacaaagag cagacgaagt tttggcgaaa
     1261 aacatcaagg cgcctgatac gaatgcattt gcaataacaa ttgcgatatt taatattgtt
     1321 tatgaagctg tttgacttca aaacacacaa aaaaaaaaat aaaacaaatt atttgaaaga
     1381 gaattaggaa tcggacgctt atcgttaggg taacaacaag aaatgcttac tgagtcacag
     1441 cctctggaaa actgccgcaa gccagagaga gagagaaaaa gagggagagc agcttagacc
     1501 gcatgtgctt gtgtgtgagg cgtctctctc ttcgtctctg ttgcgcaaac gcatagactg
     1561 cactgaaaaa atcgattacc tattttttat gaatgaatat ttgcactatt actattcaaa
     1621 actattaaga tagcaatcac attcaatagc caaatactat accacctgag cgatgcaacg
     1681 aaatgatcaa tttgagcaaa aatgctgcat atttaggacg gcatcattat agaaatgctt
     1741 cttgctgtgt acttttctct cgtctggcag ctgtttcgcc gttattgtta aaaccggctt
     1801 aagttaggtg tgttttctac gactagtgaa tgccctacta gaagatgtgt gttgcacaaa
     1861 atgtccctgg aataaccaat ttgaagtgca gatagcagta aacgtaagct aatatgaata
     1921 ttatttaact gtaatgtttt aatatcgctg gacattacta ataaacccac tataaacaca
     1981 tgtacatatg tatgttttgg catacaatga gtagttgggg aaaaaatgtg taaaagcacc
     2041 gtgaccatca cagcataaag ataaccagct gaagtatcga atatgagtaa cccccaaatt
     2101 gaatcacatg ccgcaactga taggacccat ggaagtacac tcttcatggc gatatacaag
     2161 acacacacaa gcacgaacac ccagttgcgg aggaaattct ccgtaaatga aaacccaatc
     2221 ggcgaacaat tcatacccat atatggtaaa agttttgaac gcgacttgag agcggagagc
     2281 attgcggctg ataaggtttt agcgctaagc gggctttata aaacgggctg cgggaccagt
     2341 tttcatatca ctaccgtttg agttcttgtg ctgtgtggat actcctcccg acacaaagcc
     2401 gctccatcag ccagcagtcg tctaatccag agaccccgga tcggggtacc tactagtcca
     2461 gtgtggtgga attctctaga gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2521 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2581 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2641 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2701 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2761 gctacgtcca ggagcgcacc atcttcttca agggcgacgg caactacaag acccgcgccg
     2821 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2881 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2941 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     3001 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3061 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     3121 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3181 tcggcatgga cgagctgtac aagcttgagg gcagaggaag tcttctaaca tgcggtgacg
     3241 tggaggagaa tcccggccct gctagcggta gcggcagcgg tagcatgacc gagtacaagc
     3301 ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg
     3361 cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg
     3421 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     3481 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     3541 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     3601 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     3661 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     3721 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     3781 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     3841 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgactcgag ggatccagac
     3901 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     3961 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     4021 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     4081 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcagtcgacc
     4141 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     4201 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     4261 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     4321 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     4381 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta
     4441 cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc
     4501 ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga
     4561 ccatcaggga cagcttcaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4621 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4681 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4741 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4801 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4861 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4921 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4981 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5041 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5101 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5161 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5221 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5281 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5341 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5401 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5461 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5521 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5581 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5641 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5701 tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5761 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5821 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5881 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5941 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6001 aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6061 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6121 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6181 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6241 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6301 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6361 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     6421 taggcgtatc acgaggccct ttcgtcttca agaattaatt ctatattcta aaaacacaaa
     6481 tgatacttct aaaaaaaaat catgaatggc atcaactctg aatcaaatct ttgcagatgc
     6541 acctacttct catttccact gtcacatcat ttttcc
//
