LOCUS       sequence-111229-        4560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NeoR containing 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 4560)
  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 4560)
  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-­mCh-­Poly(A)‐NeoR
FEATURES             Location/Qualifiers
     source          1..4560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        62..561
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(97..120)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     494..513
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             579..1373
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             579..1373
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1561..1580)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1605..1652
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1823..1860
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             1863..2567
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2005..2023)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2273..2292
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      2613..2622
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2675..2809
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2699..2718)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2753..2772
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2945..2992
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3011..3814
                     /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"
     rep_origin      complement(3943..4531)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4023..4042)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatgccagct cattcctccc actcatgatc tatagatccc ccgggctgca ggaattctac
       61 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg
      121 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg
      181 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
      241 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
      301 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
      361 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
      421 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
      481 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
      541 tcctcatctc cgggcctttc gaccgatcat caagcttaat gattgaacaa gatggattgc
      601 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
      661 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
      721 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
      781 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
      841 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
      901 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
      961 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     1021 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     1081 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     1141 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     1201 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     1261 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     1321 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     1381 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     1441 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     1501 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     1561 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     1621 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     1681 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     1741 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     1801 ggcaagctta catcgagatc ccggcttgtc gacgacggcg gtctccgtcg tcaggatcat
     1861 ccgtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     1921 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     1981 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     2041 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     2101 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     2161 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     2221 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     2281 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     2341 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     2401 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     2461 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     2521 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagagc ggcctgagga
     2581 gcagagccca ggcgagcaac agcgccgtgg acgccaccat gggcgatcgc ccgggaattg
     2641 actagtgcgg ccgcagttaa cgaattctct agaggatcca gacatgataa gatacattga
     2701 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     2761 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     2821 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     2881 aaacctctac aaatgtggta tggctgatta tgatcctgca agcctcgtcg ccgcggttta
     2941 ttctgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     3001 gaactaaacc atgggatcgg ccattgaaca agatggattg cacgcaggtt ctccggccgc
     3061 ttgggtggag aggctattcg gctatgactg ggcacaacag acgatcggct gctctgatgc
     3121 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3181 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3241 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3301 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     3361 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     3421 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3481 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     3541 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     3601 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     3661 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     3721 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gcccccgatt cgcagcgcat
     3781 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaataaaga
     3841 ccgaccaagc gacgtctgag agctccctgg cgaattcggt accaataaaa gagctttatt
     3901 ttcatgatct gtgtgttggt ttttggccgc gttgctggcg tttttccata ggctccgccc
     3961 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4021 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4081 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4141 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4201 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4261 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4321 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4381 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4441 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4501 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctagtgtgcg
//
