LOCUS       sequence-447850-        5214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid serves as a template for homologous DNA recombination 
            at the LIG4 gene to knock out the DNA ligase 4 protein using the 
            neomycin SelectRepair knockout approach..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5214)
  AUTHORS   Cortazar MA, Jagannathan S
  TITLE     SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through 
            Selectable Homology-Directed DNA Repair.
  JOURNAL   Methods Mol Biol. 2025;2863:397-417. doi: 
            10.1007/978-1-0716-4176-7_23.
  PUBMED    39535722
REFERENCE   2  (bases 1 to 5214)
  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: "Methods Mol
            Biol."; date: "2025"; volume: "2863:397-417. doi"; pages: " 
            10.1007/978-1-0716-4176-7_23"
COMMENT     Sequence Label: LIG4 HAL-T2A-NEO-SV40pA-HAR
FEATURES             Location/Qualifiers
     source          1..5214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             869..922
                     /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             947..1738
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             947..1738
                     /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="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(998..1017)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1608..1627
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1746..1880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1770..1789)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1824..1843
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3050..3066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3050..3066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3063..3085)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3238..3826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3318..3337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3943..3967)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4067..4858,4859..4927))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4690..4709
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4928..5032)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5131..5153
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5145..5162
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5146..5162
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 acagttatta aatgtagagg ttgaaaagat acatcattta gagcattgat ctttttttta
       61 gtaaacatta ataaacattt attatgatct cagtgatctt tgtaagatct ttacattaaa
      121 tatttagttg aatcttatct tttacttaaa gttattctct ttttgcttta ctagttaaac
      181 gagaagattc atcaccgctt tgatggctgc ctcacaaact tcacaaactg ttgcatctca
      241 cgttcctttt gcagatttgt gttcaacttt agaacgaata cagaaaagta aaggacgtgc
      301 agaaaaaatc agacacttca gggaattttt agattcttgg agaaaatttc atgatgctct
      361 tcataagaac cacaaagatg tcacagactc tttttatcca gcaatgagac taattcttcc
      421 tcagctagaa agagagagaa tggcctatgg aattaaagaa actatgcttg ctaagcttta
      481 tattgagttg cttaatttac ctagagatgg aaaagatgcc ctcaaacttt taaactacag
      541 aacacccact ggaactcatg gagatgctgg agactttgca atgattgcat attttgtgtt
      601 gaagccaaga tgtttacaga aaggaagttt aaccatacag caagtaaacg accttttaga
      661 ctcaattgcc agcaataatt ctgctaaaag aaaagaccta ataaaaaaga gccttcttca
      721 acttataact cagagttcag cacttgagca aaagtggctt atacggatga tcataaagga
      781 tttaaagctt ggtgttagtc agcaaactat cttttctgtt tttcataatg atgctgctga
      841 gttgcataat gtcactacag ccaagcttga gggcagagga agtcttctaa catgcggtga
      901 cgtggaggag aatcccggcc ctgctagcgg tagcggcagc ggtagcattg aacaagatgg
      961 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     1021 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     1081 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     1141 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     1201 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     1261 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     1321 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     1381 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     1441 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     1501 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     1561 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     1621 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     1681 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgact
     1741 cgagggatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     1801 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     1861 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     1921 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
     1981 atgatcagtc gacatctgga aaaagtctgt aggcaactgc atgatccttc tgtaggactc
     2041 agtgatattt ctatcacttt attttctgca tttaaaccaa tgctagctgc tattgcagat
     2101 attgagcaca ttgagaagga tatgaaacat cagagtttct acatagaaac caagctagat
     2161 ggtgaacgta tgcaaatgca caaagatgga gatgtatata aatacttctc tcgaaatgga
     2221 tataactaca ctgatcagtt tggtgcttct cctactgaag gttctcttac cccattcatt
     2281 cataatgcat tcaaagcaga tatacaaatc tgtattcttg atggtgagat gatggcctat
     2341 aatcctaata cacaaacttt catgcaaaag ggaactaagt ttgatattaa aagaatggta
     2401 gaggattctg atctgcaaac ttgttattgt gtttttgatg tattgatggt taataataaa
     2461 aagctagggc atgagactct gagaaagagg tatgagattc ttagtagtat ttttacacca
     2521 attccaggta gaatagaaat agtgcagaaa acacaagctc atactaagaa tgaagtaatt
     2581 gatgcattga atgaagcaat agataaaaga gaagagggaa ttatggtaaa acaacctcta
     2641 tccatctaca agccagacaa aagaggtgaa gggtggttaa aaattaaacc agagtatgtc
     2701 agtggactaa tggatgaatt ggacatttta attgttggag gatattgggg taaaggatca
     2761 cggggtggaa tgatgtctca ttttctgtgt gcagtagcag agaagccccc tcctggtgag
     2821 aagccatctg tgtttcatac tctctctcgt gttgggtctg gctgcaccat gaaagaactg
     2881 tatgatctgg gtttgaaatt ggccaagtat tggaagcctt ttcatagaaa agctccacca
     2941 agcagcattt tatgtggaac agagaagcca gaagtataca ttgaaccttg taaaggctag
     3001 gtggaggctc agtgatgata agtctgcgat ggtggatgca tgtgtcatgg tcatagctgt
     3061 ttcctgtgtg aaattgttat ccgctcagag ggcacaatcc tattccgcgc tatccgacaa
     3121 tctccaagac attaggtgga gttcagttcg gcgtatggca tatgtcgctg gaaagaacat
     3181 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3241 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3301 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3361 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3421 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3481 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3541 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3601 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3661 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3721 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3781 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3841 cttttctacg gggtctgacg ctctattcaa caaagccgcc gtcccgtcaa gtcagcgtaa
     3901 atgggtaggg ggcttcaaat cgtcctcgtg ataccaattc ggagcctgct tttttgtaca
     3961 aacttgttga taatggcaat tcaaggatct tcacctagat ccttttaaat taaaaatgaa
     4021 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4081 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4141 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4201 taccgcgaga gccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4261 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4321 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4381 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4441 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4501 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4561 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4621 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4681 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4741 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4801 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4861 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4921 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4981 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5041 cccgaaaagt gccagatacc tgaaacaaaa cccatcgtac ggccaaggaa gtctccaata
     5101 actgtgatcc accacaagcg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     5161 gtcatgcata atccgcacgc atctggaata aggaagtgcc attccgcctg acct
//
