LOCUS       sequence-410192-        7395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enables expression of 2 ORFs (with or without tags) and selection 
            with puromycin in insect cells. Includes 
            Flag-mCherry-T2A-GFP-T2A-puro with Actin5C promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7395)
  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 7395)
  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-STABLE2-puro
FEATURES             Location/Qualifiers
     source          1..7395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      2475..2484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2484..2507
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2656..2674)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             3228..3281
                     /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"
     regulatory      3300..3309
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3306..3308,3309..3311,3312..4022)
                     /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(3351..3372)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3959..3980
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4026..4079
                     /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             4104..4703
                     /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(4104..4123)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4600..4620
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4711..4845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4735..4754)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4789..4808
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(5446..6034)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5526..5545)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6205..6996,6997..7065))
                     /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     6828..6847
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7066..7170)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7238..7256
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(7320..7395,1..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 attcgccacc atggactaca aagacgatga cgacaagggc agcgtgtcca
     2521 agggcgaaga ggacaacatg gccatcatca aggagttcat gcggttcaag gtgcacatgg
     2581 aaggcagcgt gaacggccac gagttcgaga tcgagggcga gggcgaaggc aggccctacg
     2641 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg acctctgccc ttcgcctggg
     2701 acatcctgag cccccagttc atgtacggca gcaaggccta cgtgaagcac cccgccgaca
     2761 tccccgacta cctgaagctg tccttccccg agggattcaa atgggagcgg gtgatgaact
     2821 tcgaggacgg cggcgtggtg accgtgaccc aggacagcag cctgcaggac ggcgagttca
     2881 tctacaaggt gaaactgcgg ggcaccaact tccccagcga cggccccgtg atgcagaaaa
     2941 agaccatggg ctgggaggcc agcagcgagc ggatgtaccc cgaggatggc gccctgaagg
     3001 gcgagatcaa gcagcggctg aagctgaagg atggcggcca ctacgacgcc gaggtgaaaa
     3061 ccacctacaa ggccaagaaa cccgtgcagc tgcctggcgc ctacaacgtg aacatcaagc
     3121 tggacatcac cagccacaac gaggactaca ccatcgtgga gcagtacgag cgggccgagg
     3181 gcagacacag caccggcggc atggacgagc tgtataaagg cggccgcgaa ggacgcggca
     3241 gcctactgac ttgcggagat gtcgaagaga accctggccc tggttccgat atctctagag
     3301 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     3361 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     3421 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     3481 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     3541 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     3601 tcttcttcaa gggcgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     3661 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     3721 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     3781 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     3841 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3901 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     3961 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     4021 agcttgaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggccctg
     4081 ctagcggtag cggcagcggt agcatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     4141 gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     4201 cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     4261 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     4321 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     4381 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     4441 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     4501 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     4561 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     4621 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     4681 tgacccgcaa gcccggtgcc tgactcgagg gatccagaca tgataagata cattgatgag
     4741 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     4801 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4861 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     4921 ctctacaaat gtggtatggc tgattatgat cagtcgaccg atgcccttga gagccttcaa
     4981 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     5041 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     5101 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     5161 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     5221 ggccattatc gccggcatgg cggccgacgc gctgggctac gtcttgctgg cgttcgcgac
     5281 gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca tcgggatgcc
     5341 cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac agcttcaagg
     5401 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5461 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5521 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5581 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5641 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5701 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5761 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5821 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5881 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5941 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6001 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6061 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6121 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6181 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6241 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6301 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6361 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6421 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6481 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg
     6541 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6601 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6661 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6721 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6781 atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc
     6841 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6901 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6961 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7021 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7081 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7141 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7201 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7261 tcgtcttcaa gaattaattc tatattctaa aaacacaaat gatacttcta aaaaaaaatc
     7321 atgaatggca tcaactctga atcaaatctt tgcagatgca cctacttctc atttccactg
     7381 tcacatcatt tttcc
//
