LOCUS       sequence-410195-        7194 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Enables expression of 2 ORFs (with or without tags) and selection 
            with blasticidin in insect cells. Includes 
            Flag-mCherry-T2A-GFP-T2A-blast with Actin5C promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7194)
  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 7194)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-blast
FEATURES             Location/Qualifiers
     source          1..7194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      31..40
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             40..63
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(212..230)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             784..837
                     /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      856..865
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(862..864,865..867,868..1578)
                     /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(907..928)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1515..1536
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1582..1635
                     /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             1660..2058
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    2066..2200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2090..2109)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2144..2163
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(2801..3389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2881..2900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3560..4351,4352..4420))
                     /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     4183..4202
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4421..4525)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4593..4611
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4675..7184
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
ORIGIN
        1 ggtacctact agtccagtgt ggtggaattc gccaccatgg actacaaaga cgatgacgac
       61 aagggcagcg tgtccaaggg cgaagaggac aacatggcca tcatcaagga gttcatgcgg
      121 ttcaaggtgc acatggaagg cagcgtgaac ggccacgagt tcgagatcga gggcgagggc
      181 gaaggcaggc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggacct
      241 ctgcccttcg cctgggacat cctgagcccc cagttcatgt acggcagcaa ggcctacgtg
      301 aagcaccccg ccgacatccc cgactacctg aagctgtcct tccccgaggg attcaaatgg
      361 gagcgggtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga cagcagcctg
      421 caggacggcg agttcatcta caaggtgaaa ctgcggggca ccaacttccc cagcgacggc
      481 cccgtgatgc agaaaaagac catgggctgg gaggccagca gcgagcggat gtaccccgag
      541 gatggcgccc tgaagggcga gatcaagcag cggctgaagc tgaaggatgg cggccactac
      601 gacgccgagg tgaaaaccac ctacaaggcc aagaaacccg tgcagctgcc tggcgcctac
      661 aacgtgaaca tcaagctgga catcaccagc cacaacgagg actacaccat cgtggagcag
      721 tacgagcggg ccgagggcag acacagcacc ggcggcatgg acgagctgta taaaggcggc
      781 cgcgaaggac gcggcagcct actgacttgc ggagatgtcg aagagaaccc tggccctggt
      841 tccgatatct ctagagccac catggtgagc aagggcgagg agctgttcac cggggtggtg
      901 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      961 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1021 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     1081 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1141 gtccaggagc gcaccatctt cttcaagggc gacggcaact acaagacccg cgccgaggtg
     1201 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1261 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1321 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1381 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1441 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     1501 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1561 atggacgagc tgtacaagct tgagggcaga ggaagtcttc taacatgcgg tgacgtggag
     1621 gagaatcccg gccctgctag cggtagcggc agcggtagca tggccaagcc tttgtctcaa
     1681 gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa
     1741 gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat
     1801 gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct
     1861 gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg
     1921 agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata
     1981 gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt
     2041 tatgtgtggg agggctaact cgagggatcc agacatgata agatacattg atgagtttgg
     2101 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     2161 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     2221 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     2281 caaatgtggt atggctgatt atgatcagtc gaccgatgcc cttgagagcc ttcaacccag
     2341 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     2401 ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc
     2461 gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg
     2521 ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca
     2581 ttatcgccgg catggcggcc gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag
     2641 gctggatggc cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt
     2701 tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt caaggccagc
     2761 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2821 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2881 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2941 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3001 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3061 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3121 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3181 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3241 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3301 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3361 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3421 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3481 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3541 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3601 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3661 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3721 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3781 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3841 cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt
     3901 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3961 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4021 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4081 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4141 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     4201 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4261 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4321 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4381 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4441 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4501 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     4561 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     4621 ttcaagaatt aattctatat tctaaaaaca caaatgatac ttctaaaaaa aaatcatgaa
     4681 tggcatcaac tctgaatcaa atctttgcag atgcacctac ttctcatttc cactgtcaca
     4741 tcatttttcc agatctcgct gcctgttatg tggcccacaa accaagacac gttttatggc
     4801 cattaaagct ggctgatcgt cgccaaacac caaatacata atgaatatgt acacattcga
     4861 gaaagaagcg atcaaagaag cgtcttcggg cggagtagga gaatgcggag gagaaggaga
     4921 acgagctgat ctagtatctc tccacaatcc aatgccaact gaccaactgg ccatattcgg
     4981 agcaatttga agccaatttc catcgcctgg cgatcgctcc attcttggct atatgttttt
     5041 caccgttacc cggggccatt ttcaaagact cgtcggcaag ataagattgt gtcactcgct
     5101 gtctctcttc atttgtcgaa gaatgctgag gaatttcgcg atgacgtcgg cgagtatttt
     5161 gaagaatgag aataatttgt atttatacga aaatcagtta gtggaatttt ctacaaaaac
     5221 atgttatcta tagataattt tgttgcaaaa tatgttgact atgacaaaga ttgtatgtat
     5281 atacctttaa tgtattctca ttttcttatg tatttataat ggcaatgatg atactgatga
     5341 tattttaaga tgatgccaga ccaaaaggct tgaatttctg cgtcttttgc cgaacgcagt
     5401 gcatgtgcaa ttgttgtttt ttggaatatt caattttcgg actgtccgct ttgatttcag
     5461 tttcttggct tattcaaaaa gcaaagtaaa gccaaaaaag cgagatggca ataccaaatg
     5521 cggcaaaacg gtagtggaag gaaaggggtg cggggcagcg gaaggaaggg tggggcgggg
     5581 cgtggcgggg tctgtggctg ggcgcgacgt caccgacgtt ggagccactc ctttgaccat
     5641 gtgtgcgtgt gtgtattatt cgtgtctcgc cactcgccgg ttgttttttt ctttttatgc
     5701 tgcgctctct ctagcgccat ctcgcttacg catgctcaac gcaccgcatg ttgccgtttc
     5761 cttttatgcg tcattttggc tcgaaatagg caattattta aacaaagatt agtcaacgaa
     5821 aacgctaaaa taaataagtc tacaatatgg ttacttattg ccatgtgtgt gcagccaacg
     5881 atagcaacaa aagcaacaac acaggtggct ttccctcttt cactttttgt ttgcaagccg
     5941 cgtgcgagca agacggcacg accggcaaac gcaattacgc tgacaaagag cagacgaagt
     6001 tttggcgaaa aacatcaagg cgcctgatac gaatgcattt gcaataacaa ttgcgatatt
     6061 taatattgtt tatgaagctg tttgacttca aaacacacaa aaaaaaaaat aaaacaaatt
     6121 atttgaaaga gaattaggaa tcggacgctt atcgttaggg taacaacaag aaatgcttac
     6181 tgagtcacag cctctggaaa actgccgcaa gccagagaga gagagaaaaa gagggagagc
     6241 agcttagacc gcatgtgctt gtgtgtgagg cgtctctctc ttcgtctctg ttgcgcaaac
     6301 gcatagactg cactgaaaaa atcgattacc tattttttat gaatgaatat ttgcactatt
     6361 actattcaaa actattaaga tagcaatcac attcaatagc caaatactat accacctgag
     6421 cgatgcaacg aaatgatcaa tttgagcaaa aatgctgcat atttaggacg gcatcattat
     6481 agaaatgctt cttgctgtgt acttttctct cgtctggcag ctgtttcgcc gttattgtta
     6541 aaaccggctt aagttaggtg tgttttctac gactagtgaa tgccctacta gaagatgtgt
     6601 gttgcacaaa atgtccctgg aataaccaat ttgaagtgca gatagcagta aacgtaagct
     6661 aatatgaata ttatttaact gtaatgtttt aatatcgctg gacattacta ataaacccac
     6721 tataaacaca tgtacatatg tatgttttgg catacaatga gtagttgggg aaaaaatgtg
     6781 taaaagcacc gtgaccatca cagcataaag ataaccagct gaagtatcga atatgagtaa
     6841 cccccaaatt gaatcacatg ccgcaactga taggacccat ggaagtacac tcttcatggc
     6901 gatatacaag acacacacaa gcacgaacac ccagttgcgg aggaaattct ccgtaaatga
     6961 aaacccaatc ggcgaacaat tcatacccat atatggtaaa agttttgaac gcgacttgag
     7021 agcggagagc attgcggctg ataaggtttt agcgctaagc gggctttata aaacgggctg
     7081 cgggaccagt tttcatatca ctaccgtttg agttcttgtg ctgtgtggat actcctcccg
     7141 acacaaagcc gctccatcag ccagcagtcg tctaatccag agaccccgga tcgg
//
