LOCUS       sequence-122031-        2809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the generation of GFP-knockin fusions via Bxb1-mediated 
            recombination into MIN-tagged cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2809)
  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 2809)
  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
FEATURES             Location/Qualifiers
     source          1..2809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..40
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(398..431)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        437..484
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             503..1306
                     /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
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(566..585)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1176..1195
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(1435..2023)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1515..1534)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(2058..2091)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2155..2173
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2155..2172
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2225..2244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2663..2686
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2726..2770
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2783..2803
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 ccggcttgtc gacgacggcg gtctccgtcg tcaggatcat ccgtgtccaa gggcgaggaa
       61 ctgttcaccg gcgtggtgcc catcctggtg gagctggacg gcgacgtgaa cggccacaag
      121 ttcagcgtgt ccggcgaggg cgaaggcgac gccacctacg gcaagctgac cctgaagttc
      181 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tggtgaccac cctgacctac
      241 ggcgtgcagt gcttcagcag ataccccgac cacatgaagc agcacgattt cttcaagagc
      301 gccatgcccg agggctacgt ccaggtaagt gtgtacggtg ggatccggtc tatataagga
      361 gagctgtctg caaaccctat gctactgcgg ccgctcgata acttcgtata gcatacatta
      421 tacgaagtta tattctgttg acaattaatc atcggcatag tatatcggca tagtataata
      481 cgacaaggtg aggaactaaa ccatgggatc ggccattgaa caagatggat tgcacgcagg
      541 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacgatcgg
      601 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
      661 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
      721 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
      781 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
      841 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
      901 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
      961 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     1021 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     1081 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     1141 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     1201 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgcccccga
     1261 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     1321 ttcgaataaa gaccgaccaa gcgacgtctg agagctccct ggcgaattcg gtaccaataa
     1381 aagagcttta ttttcatgat ctgtgtgttg gtttttggcc gcgttgctgg cgtttttcca
     1441 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1501 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1561 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1621 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1681 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1741 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1801 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1861 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1921 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1981 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2041 ttctagtgtg cggcgcgata acttcgtata gcatacatta tacgaagtta tcgctcgagt
     2101 aggacaaatc cgccgggata attaatacat aaccttatgt atcatacaca tacgatttag
     2161 gtgacactat agaagcttaa catccacttt tttttcccca caggagcgca ccatcttttt
     2221 caaggacgac ggcaactaca agaccagggc cgaggtgaag ttcgagggcg acacactggt
     2281 gaacaggatc gagctgaagg gcatcgactt caaagaggac ggcaacatcc tgggccacaa
     2341 gctggaatac aactacaaca gccacaacgt gtacattatg gccgacaagc agaagaacgg
     2401 catcaaggtc aacttcaaga tccggcacaa catcgaggac ggcagcgtgc agctggccga
     2461 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2521 cctgagcacc cagagcgctc tcagcaagga ccccaacgag aagcgggacc acatggtgct
     2581 gaaagaattt gtgacagccg ccggaatcac cctgggcatg gacgagctgt acaagagcag
     2641 cggccccagc ggcagcagca gcctggaagt gctgttccag ggccccctga gcagctctgg
     2701 cccctctggc agcgccgcca agttcaaaga aacagctgct gctaaattcg agcggcagca
     2761 catggacagc ggaggaggag gcgagaacct gtactttcag ggaggcgga
//
