LOCUS       sequence-410194-        6375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enables expression of an ORF (with or without tags) and selection 
            with blasticidin in insect cells. Includes GFP-T2A-blast with 
            Actin5C promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6375)
  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 6375)
  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-blast
FEATURES             Location/Qualifiers
     source          1..6375
                     /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..3683
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3691..3825
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3715..3734)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3769..3788
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(4426..5014)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4506..4525)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5185..5976,5977..6045))
                     /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     5808..5827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6046..6150)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6218..6236
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(6300..6375,1..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 tagcatggcc aagcctttgt
     3301 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
     3361 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
     3421 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
     3481 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
     3541 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag
     3601 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
     3661 ctggttatgt gtgggagggc taactcgagg gatccagaca tgataagata cattgatgag
     3721 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     3781 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     3841 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     3901 ctctacaaat gtggtatggc tgattatgat cagtcgaccg atgcccttga gagccttcaa
     3961 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     4021 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     4081 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     4141 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     4201 ggccattatc gccggcatgg cggccgacgc gctgggctac gtcttgctgg cgttcgcgac
     4261 gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca tcgggatgcc
     4321 cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac agcttcaagg
     4381 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4441 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4501 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4561 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4621 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4681 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4741 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4801 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4861 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4921 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4981 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5041 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5101 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5161 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5221 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5281 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5341 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5401 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5461 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg
     5521 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5581 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5641 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5701 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5761 atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc
     5821 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5881 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5941 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6001 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6061 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6121 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6181 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     6241 tcgtcttcaa gaattaattc tatattctaa aaacacaaat gatacttcta aaaaaaaatc
     6301 atgaatggca tcaactctga atcaaatctt tgcagatgca cctacttctc atttccactg
     6361 tcacatcatt tttcc
//
