LOCUS       sequence-447851-        5445 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 
            hygromycin B SelectRepair knockout approach..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5445)
  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 5445)
  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-HYG-SV40pA-HAR
FEATURES             Location/Qualifiers
     source          1..5445
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(570..623)
                     /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"
     primer_bind     complement(1544..1561)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1544..1560)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1553..1575)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        1674..1778
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1779..1847,1848..2639)
                     /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     complement(1997..2016)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    2739..2763
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     rep_origin      2880..3468
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3369..3388
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3621..3643
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3640..3656
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3640..3656
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4826..4960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4863..4882)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4917..4936
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4968..5445,1..545))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(join(4968..5445,1..545))
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
ORIGIN
        1 agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt
       61 tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat
      121 tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca
      181 gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc
      241 aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgctg
      301 aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa
      361 acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc
      421 cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg
      481 gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc
      541 tttttgctac cgctgccgct accgctagca gggccgggat tctcctccac gtcaccgcat
      601 gttagaagac ttcctctgcc ctcaagcttg gctgtagtga cattatgcaa ctcagcagca
      661 tcattatgaa aaacagaaaa gatagtttgc tgactaacac caagctttaa atcctttatg
      721 atcatccgta taagccactt ttgctcaagt gctgaactct gagttataag ttgaagaagg
      781 ctctttttta ttaggtcttt tcttttagca gaattattgc tggcaattga gtctaaaagg
      841 tcgtttactt gctgtatggt taaacttcct ttctgtaaac atcttggctt caacacaaaa
      901 tatgcaatca ttgcaaagtc tccagcatct ccatgagttc cagtgggtgt tctgtagttt
      961 aaaagtttga gggcatcttt tccatctcta ggtaaattaa gcaactcaat ataaagctta
     1021 gcaagcatag tttctttaat tccataggcc attctctctc tttctagctg aggaagaatt
     1081 agtctcattg ctggataaaa agagtctgtg acatctttgt ggttcttatg aagagcatca
     1141 tgaaattttc tccaagaatc taaaaattcc ctgaagtgtc tgattttttc tgcacgtcct
     1201 ttacttttct gtattcgttc taaagttgaa cacaaatctg caaaaggaac gtgagatgca
     1261 acagtttgtg aagtttgtga ggcagccatc aaagcggtga tgaatcttct cgtttaacta
     1321 gtaaagcaaa aagagaataa ctttaagtaa aagataagat tcaactaaat atttaatgta
     1381 aagatcttac aaagatcact gagatcataa taaatgttta ttaatgttta ctaaaaaaaa
     1441 gatcaatgct ctaaatgatg tatcttttca acctctacat ttaataactg taggtcaggc
     1501 ggaatggcac ttccttattc cagatgcgtg cggattatgc atgactggcc gtcgttttac
     1561 aacgtcgtga ctgggaaaac cctggcgctt gtggtggatc acagttattg gagacttcct
     1621 tggccgtacg atgggttttg tttcaggtat ctggcacttt tcggggaaat gtgcgcggaa
     1681 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1741 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1801 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1861 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1921 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1981 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     2041 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     2101 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2161 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2221 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2281 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     2341 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     2401 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2461 ttattgctga taaatctgga gccggtgagc gtggctctcg cggtatcatt gcagcactgg
     2521 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2581 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2641 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2701 aaaggatcta ggtgaagatc cttgaattgc cattatcaac aagtttgtac aaaaaagcag
     2761 gctccgaatt ggtatcacga ggacgatttg aagcccccta cccatttacg ctgacttgac
     2821 gggacggcgg ctttgttgaa tagagcgtca gaccccgtag aaaagatcaa aggatcttct
     2881 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2941 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3001 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3061 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3121 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3181 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3241 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3301 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3361 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3421 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3481 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttccagcg
     3541 acatatgcca tacgccgaac tgaactccac ctaatgtctt ggagattgtc ggatagcgcg
     3601 gaataggatt gtgccctctg agcggataac aatttcacac aggaaacagc tatgaccatg
     3661 acacatgcat ccaccatcgc agacttatca tcactgagcc tccacctagc ctttacaagg
     3721 ttcaatgtat acttctggct tctctgttcc acataaaatg ctgcttggtg gagcttttct
     3781 atgaaaaggc ttccaatact tggccaattt caaacccaga tcatacagtt ctttcatggt
     3841 gcagccagac ccaacacgag agagagtatg aaacacagat ggcttctcac caggaggggg
     3901 cttctctgct actgcacaca gaaaatgaga catcattcca ccccgtgatc ctttacccca
     3961 atatcctcca acaattaaaa tgtccaattc atccattagt ccactgacat actctggttt
     4021 aatttttaac cacccttcac ctcttttgtc tggcttgtag atggatagag gttgttttac
     4081 cataattccc tcttctcttt tatctattgc ttcattcaat gcatcaatta cttcattctt
     4141 agtatgagct tgtgttttct gcactatttc tattctacct ggaattggtg taaaaatact
     4201 actaagaatc tcatacctct ttctcagagt ctcatgccct agctttttat tattaaccat
     4261 caatacatca aaaacacaat aacaagtttg cagatcagaa tcctctacca ttcttttaat
     4321 atcaaactta gttccctttt gcatgaaagt ttgtgtatta ggattatagg ccatcatctc
     4381 accatcaaga atacagattt gtatatctgc tttgaatgca ttatgaatga atggggtaag
     4441 agaaccttca gtaggagaag caccaaactg atcagtgtag ttatatccat ttcgagagaa
     4501 gtatttatat acatctccat ctttgtgcat ttgcatacgt tcaccatcta gcttggtttc
     4561 tatgtagaaa ctctgatgtt tcatatcctt ctcaatgtgc tcaatatctg caatagcagc
     4621 tagcattggt ttaaatgcag aaaataaagt gatagaaata tcactgagtc ctacagaagg
     4681 atcatgcagt tgcctacaga ctttttccag atgtcgactg atcataatca gccataccac
     4741 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     4801 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     4861 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4921 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cctcgagcta ttcctttgcc
     4981 ctcggacgag tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg
     5041 gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat
     5101 cggacgattg cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc
     5161 aagctctgat agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat
     5221 cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac
     5281 cacggcctcc agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg
     5341 ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa
     5401 tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg cccaa
//
