LOCUS       sequence-228406-        6762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6762)
  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 6762)
  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-neo
FEATURES             Location/Qualifiers
     source          1..6762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..33
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        97..2606
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     regulatory      2653..2662
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2659..2661,2662..2664,2665..3375)
                     /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(2704..2725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3312..3333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3379..3432
                     /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             3439..4242
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3502..3521)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4112..4131
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4250..4384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4274..4293)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4328..4347
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(4985..5573)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5065..5084)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5744..6535,6536..6604))
                     /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     6367..6386
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6605..6709)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa ttaattctat
       61 attctaaaaa cacaaatgat acttctaaaa aaaaatcatg aatggcatca actctgaatc
      121 aaatctttgc agatgcacct acttctcatt tccactgtca catcattttt ccagatctcg
      181 ctgcctgtta tgtggcccac aaaccaagac acgttttatg gccattaaag ctggctgatc
      241 gtcgccaaac accaaataca taatgaatat gtacacattc gagaaagaag cgatcaaaga
      301 agcgtcttcg ggcggagtag gagaatgcgg aggagaagga gaacgagctg atctagtatc
      361 tctccacaat ccaatgccaa ctgaccaact ggccatattc ggagcaattt gaagccaatt
      421 tccatcgcct ggcgatcgct ccattcttgg ctatatgttt ttcaccgtta cccggggcca
      481 ttttcaaaga ctcgtcggca agataagatt gtgtcactcg ctgtctctct tcatttgtcg
      541 aagaatgctg aggaatttcg cgatgacgtc ggcgagtatt ttgaagaatg agaataattt
      601 gtatttatac gaaaatcagt tagtggaatt ttctacaaaa acatgttatc tatagataat
      661 tttgttgcaa aatatgttga ctatgacaaa gattgtatgt atataccttt aatgtattct
      721 cattttctta tgtatttata atggcaatga tgatactgat gatattttaa gatgatgcca
      781 gaccaaaagg cttgaatttc tgcgtctttt gccgaacgca gtgcatgtgc aattgttgtt
      841 ttttggaata ttcaattttc ggactgtccg ctttgatttc agtttcttgg cttattcaaa
      901 aagcaaagta aagccaaaaa agcgagatgg caataccaaa tgcggcaaaa cggtagtgga
      961 aggaaagggg tgcggggcag cggaaggaag ggtggggcgg ggcgtggcgg ggtctgtggc
     1021 tgggcgcgac gtcaccgacg ttggagccac tcctttgacc atgtgtgcgt gtgtgtatta
     1081 ttcgtgtctc gccactcgcc ggttgttttt ttctttttat gctgcgctct ctctagcgcc
     1141 atctcgctta cgcatgctca acgcaccgca tgttgccgtt tccttttatg cgtcattttg
     1201 gctcgaaata ggcaattatt taaacaaaga ttagtcaacg aaaacgctaa aataaataag
     1261 tctacaatat ggttacttat tgccatgtgt gtgcagccaa cgatagcaac aaaagcaaca
     1321 acacaggtgg ctttccctct ttcacttttt gtttgcaagc cgcgtgcgag caagacggca
     1381 cgaccggcaa acgcaattac gctgacaaag agcagacgaa gttttggcga aaaacatcaa
     1441 ggcgcctgat acgaatgcat ttgcaataac aattgcgata tttaatattg tttatgaagc
     1501 tgtttgactt caaaacacac aaaaaaaaaa ataaaacaaa ttatttgaaa gagaattagg
     1561 aatcggacgc ttatcgttag ggtaacaaca agaaatgctt actgagtcac agcctctgga
     1621 aaactgccgc aagccagaga gagagagaaa aagagggaga gcagcttaga ccgcatgtgc
     1681 ttgtgtgtga ggcgtctctc tcttcgtctc tgttgcgcaa acgcatagac tgcactgaaa
     1741 aaatcgatta cctatttttt atgaatgaat atttgcacta ttactattca aaactattaa
     1801 gatagcaatc acattcaata gccaaatact ataccacctg agcgatgcaa cgaaatgatc
     1861 aatttgagca aaaatgctgc atatttagga cggcatcatt atagaaatgc ttcttgctgt
     1921 gtacttttct ctcgtctggc agctgtttcg ccgttattgt taaaaccggc ttaagttagg
     1981 tgtgttttct acgactagtg aatgccctac tagaagatgt gtgttgcaca aaatgtccct
     2041 ggaataacca atttgaagtg cagatagcag taaacgtaag ctaatatgaa tattatttaa
     2101 ctgtaatgtt ttaatatcgc tggacattac taataaaccc actataaaca catgtacata
     2161 tgtatgtttt ggcatacaat gagtagttgg ggaaaaaatg tgtaaaagca ccgtgaccat
     2221 cacagcataa agataaccag ctgaagtatc gaatatgagt aacccccaaa ttgaatcaca
     2281 tgccgcaact gataggaccc atggaagtac actcttcatg gcgatataca agacacacac
     2341 aagcacgaac acccagttgc ggaggaaatt ctccgtaaat gaaaacccaa tcggcgaaca
     2401 attcataccc atatatggta aaagttttga acgcgacttg agagcggaga gcattgcggc
     2461 tgataaggtt ttagcgctaa gcgggcttta taaaacgggc tgcgggacca gttttcatat
     2521 cactaccgtt tgagttcttg tgctgtgtgg atactcctcc cgacacaaag ccgctccatc
     2581 agccagcagt cgtctaatcc agagaccccg gatcggggta cctactagtc cagtgtggtg
     2641 gaattctcta gagccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2701 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2761 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2821 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2881 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2941 caggagcgca ccatcttctt caagggcgac ggcaactaca agacccgcgc cgaggtgaag
     3001 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     3061 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     3121 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     3181 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     3241 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     3301 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3361 gacgagctgt acaagcttga gggcagagga agtcttctaa catgcggtga cgtggaggag
     3421 aatcccggcc ctgctagcat gggatcggcc attgaacaag atggattgca cgcaggttct
     3481 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3541 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3601 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     3661 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3721 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3781 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3841 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3901 cttgtcgatc aggatgatct ggacgaagaa catcaggggc tcgcgccagc cgaactgttc
     3961 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4021 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4081 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagaa
     4141 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4201 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gactcgaggg atccagacat
     4261 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     4321 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     4381 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     4441 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc agtcgaccga
     4501 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     4561 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     4621 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     4681 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     4741 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccgacgcg ctgggctacg
     4801 tcttgctggc gttcgcgacg cgaggctgga tggccttccc cattatgatt cttctcgctt
     4861 ccggcggcat cgggatgccc gcgttgcagg ccatgctgtc caggcaggta gatgacgacc
     4921 atcagggaca gcttcaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4981 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5041 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5101 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5161 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5221 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5281 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5341 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5401 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     5461 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5521 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5581 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5641 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5701 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5761 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5821 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5881 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5941 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6001 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     6061 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6121 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6181 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6241 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6301 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6361 cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6421 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6481 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6541 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6601 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6661 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6721 gaaaagtgcc acctgacgtc taagaaacca ttattatcat ga
//
