LOCUS       sequence-122099-        2755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the generation of gene disruptions via Bxb1-mediated 
            recombination into MIN-tagged cell lines. This vector can also be 
            used to express cDNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2755)
  AUTHORS   Mulholland CB, Smets M, Schmidtmann E, Leidescher S, Markaki Y, 
            Hofweber M, Qin W, Manzo M, Kremmer E, Thanisch K, Bauer C, Rombaut 
            P, Herzog F, Leonhardt H, Bultmann S
  TITLE     A modular open platform for systematic functional studies under 
            physiological conditions.
  JOURNAL   Nucleic Acids Res. 2015 May 24. pii: gkv550.
  PUBMED    26007658
REFERENCE   2  (bases 1 to 2755)
  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: "Nucleic 
            Acids Res. 2015 May 24. pii: gkv550."
COMMENT     Sequence Label: attB-­GFP-­Poly(A)
FEATURES             Location/Qualifiers
     source          1..2755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    9..46
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             join(49..51,52..762)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(49..51,52..762)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(91..112)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(352..371)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     699..720
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      808..817
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    870..1004
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(894..913)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     948..967
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1140..1187
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1206..2009
                     /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
                     EHQGLAPAELFARLKARMPGGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1269..1288)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1879..1898
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(2138..2726)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2218..2237)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatatcccgg cttgtcgacg acggcggtct ccgtcgtcag gatcatccgt gagcaagggc
       61 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
      121 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
      181 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
      241 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
      301 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
      361 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
      421 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
      481 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
      541 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
      601 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
      661 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
      721 accgccgccg ggatcactct cggcatggac gagctgtaca agagcggcct gaggagcaga
      781 gcccaggcga gcaacagcgc cgtggacgcc accatgggcg atcgcccggg aattgactag
      841 tgcggccgca gttaacgaat tctctagagg atccagacat gataagatac attgatgagt
      901 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
      961 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     1021 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     1081 tctacaaatg tggtatggct gattatgatc ctgcaagcct cgtcgccgcg gtttattctg
     1141 ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact
     1201 aaaccatggg atcggccatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     1261 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     1321 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     1381 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     1441 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     1501 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     1561 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     1621 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     1681 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     1741 cgcgcatgcc cggcggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     1801 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     1861 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     1921 gggctgaccg cttcctcgtg ctttacggta tcgccgcccc cgattcgcag cgcatcgcct
     1981 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaat aaagaccgac
     2041 caagcgacgt ctgagagctc cctggcgaat tcggtaccaa taaaagagct ttattttcat
     2101 gatctgtgtg ttggtttttg gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2161 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2221 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2281 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2341 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2401 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2461 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2521 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2581 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2641 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2701 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctagt gtgcg
//
