LOCUS       sequence-447849-        5136 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 
            SelectRepair knockout GFP approach..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5136)
  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 5136)
  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-GFP-SV40pA-HAR
FEATURES             Location/Qualifiers
     source          1..5136
                     /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..1660
                     /codon_start=1
                     /product="monomeric green fluorescent protein, also known 
                     as mTagGFP"
                     /label=TagGFP2
                     /note="mammalian codon-optimized"
                     /translation="SGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKF
                     ICTTGKLPVPWPTLVTTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDGK
                     YKTRGEVKFEGDTLVNRIELKGKDFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEAN
                     FKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDRNEARDHMVLLES
                     FSACCHTHGMDELYR"
     polyA_signal    1668..1802
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1692..1711)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1746..1765
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2972..2988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2972..2988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2985..3007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3160..3748)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3240..3259)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3865..3889)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3989..4780,4781..4849))
                     /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     4612..4631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4850..4954)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5053..5075
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5067..5084
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5068..5084
                     /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 ggtagcagcg ggggcgagga
      961 gctgttcgcc ggcatcgtgc ccgtgctgat cgagctggac ggcgacgtgc acggccacaa
     1021 gttcagcgtg cgcggcgagg gcgagggcga cgccgactac ggcaagctgg agatcaagtt
     1081 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctggtgacca ccctctgcta
     1141 cggcatccag tgcttcgccc gctaccccga gcacatgaag atgaacgact tcttcaagag
     1201 cgccatgccc gagggctaca tccaggagcg caccatccag ttccaggacg acggcaagta
     1261 caagacccgc ggcgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1321 gggcaaggac ttcaaggagg acggcaacat cctgggccac aagctggagt acagcttcaa
     1381 cagccacaac gtgtacatcc gccccgacaa ggccaacaac ggcctggagg ctaacttcaa
     1441 gacccgccac aacatcgagg gcggcggcgt gcagctggcc gaccactacc agaccaacgt
     1501 gcccctgggc gacggccccg tgctgatccc catcaaccac tacctgagca ctcagaccaa
     1561 gatcagcaag gaccgcaacg aggcccgcga ccacatggtg ctcctggagt ccttcagcgc
     1621 ctgctgccac acccacggca tggacgagct gtacaggtaa ctcgagggat ccagacatga
     1681 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     1741 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     1801 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
     1861 tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcag tcgacatctg
     1921 gaaaaagtct gtaggcaact gcatgatcct tctgtaggac tcagtgatat ttctatcact
     1981 ttattttctg catttaaacc aatgctagct gctattgcag atattgagca cattgagaag
     2041 gatatgaaac atcagagttt ctacatagaa accaagctag atggtgaacg tatgcaaatg
     2101 cacaaagatg gagatgtata taaatacttc tctcgaaatg gatataacta cactgatcag
     2161 tttggtgctt ctcctactga aggttctctt accccattca ttcataatgc attcaaagca
     2221 gatatacaaa tctgtattct tgatggtgag atgatggcct ataatcctaa tacacaaact
     2281 ttcatgcaaa agggaactaa gtttgatatt aaaagaatgg tagaggattc tgatctgcaa
     2341 acttgttatt gtgtttttga tgtattgatg gttaataata aaaagctagg gcatgagact
     2401 ctgagaaaga ggtatgagat tcttagtagt atttttacac caattccagg tagaatagaa
     2461 atagtgcaga aaacacaagc tcatactaag aatgaagtaa ttgatgcatt gaatgaagca
     2521 atagataaaa gagaagaggg aattatggta aaacaacctc tatccatcta caagccagac
     2581 aaaagaggtg aagggtggtt aaaaattaaa ccagagtatg tcagtggact aatggatgaa
     2641 ttggacattt taattgttgg aggatattgg ggtaaaggat cacggggtgg aatgatgtct
     2701 cattttctgt gtgcagtagc agagaagccc cctcctggtg agaagccatc tgtgtttcat
     2761 actctctctc gtgttgggtc tggctgcacc atgaaagaac tgtatgatct gggtttgaaa
     2821 ttggccaagt attggaagcc ttttcataga aaagctccac caagcagcat tttatgtgga
     2881 acagagaagc cagaagtata cattgaacct tgtaaaggct aggtggaggc tcagtgatga
     2941 taagtctgcg atggtggatg catgtgtcat ggtcatagct gtttcctgtg tgaaattgtt
     3001 atccgctcag agggcacaat cctattccgc gctatccgac aatctccaag acattaggtg
     3061 gagttcagtt cggcgtatgg catatgtcgc tggaaagaac atgtgagcaa aaggccagca
     3121 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3181 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3241 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3301 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3361 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3421 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3481 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3541 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3601 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3661 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3721 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3781 cgctctattc aacaaagccg ccgtcccgtc aagtcagcgt aaatgggtag ggggcttcaa
     3841 atcgtcctcg tgataccaat tcggagcctg cttttttgta caaacttgtt gataatggca
     3901 attcaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3961 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4021 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4081 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gagccacgct
     4141 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4201 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4261 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4321 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4381 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4441 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4501 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4561 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4621 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4681 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4741 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4801 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4861 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4921 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccagata
     4981 cctgaaacaa aacccatcgt acggccaagg aagtctccaa taactgtgat ccaccacaag
     5041 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtcatgca taatccgcac
     5101 gcatctggaa taaggaagtg ccattccgcc tgacct
//
