LOCUS       sequence-432318-        8190 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Homologous recombination donor to insert halo tag at the N terminal 
            of the endogenous XRCC4 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8190)
  AUTHORS   Mikhova M, Goff NJ, Janovic T, Heyza JR, Meek K, Schmidt JC
  TITLE     Single-molecule imaging reveals the kinetics of non-homologous 
            end-joining in living cells.
  JOURNAL   Nat Commun. 2024 Nov 23;15(1):10159. doi: 
            10.1038/s41467-024-54545-y.
  PUBMED    39578493
REFERENCE   2  (bases 1 to 8190)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-11-23"; volume: "15(1):10159. doi"; pages: " 
            10.1038/s41467-024-54545-y"
COMMENT     Sequence Label: N-Terminal Halo XRCC4
FEATURES             Location/Qualifiers
     source          1..8190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     254..271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  405..629
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(696..1229)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1418..1423,1424..1440,1441..1446))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    1954..2002
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2027..2046
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(2138..2247)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2271..2362
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2287..2304
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             3050..3115
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3116..3149
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3156..3204
                     /note="synthetic polyadenylation signal"
     CDS             complement(3205..3804)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3288..3308)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3785..3804
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3811..4127)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3825..3960
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    4134..4167
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4176..5066
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             5079..5099
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     polyA_signal    5574..5708
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5611..5630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5665..5684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  5737..5902
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      6086..6540
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6173..6192)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6382..6403
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6566..6670
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6671..6739,6740..7531)
                     /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(6889..6908)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7702..8190,1..100)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
       61 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      121 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
      181 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
      241 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
      301 tctccttacg catctgtgcg gtatttcaca ccgcatagac cagccgcgta acctggcaaa
      361 atcggttacg gttgagtaat aaatggatgc cctgcgtaag cgggtgtggg cggacaataa
      421 agtcttaaac tgaacaaaat agatctaaac tatgacaata aagtcttaaa ctagacagaa
      481 tagttgtaaa ctgaaatcag tccagttatg ctgtgaaaaa gcatactgga cttttgttat
      541 ggctaaagca aactcttcat tttctgaagt gcaaattgcc cgtcgtatta aagaggggcg
      601 tggccaaggg catggtaaag actatattcg cggcgttgtg acaatttacc gaacaactcc
      661 gcggccggga agccgatctc ggcttgaacg aattgttagg tggcggtact tgggtcgata
      721 tcaaagtgca tcacttcttc ccgtatgccc aactttgtat agagagccac tgcgggatcg
      781 tcaccgtaat ctgcttgcac gtagatcaca taagcaccaa gcgcgttggc ctcatgcttg
      841 aggagattga tgagcgcggt ggcaatgccc tgcctccggt gctcgccgga gactgcgaga
      901 tcatagatat agatctcact acgcggctgc tcaaacttgg gcagaacgta agccgcgaga
      961 gcgccaacaa ccgcttcttg gtcgaaggca gcaagcgcga tgaatgtctt actacggagc
     1021 aagttcccga ggtaatcgga gtccggctga tgttgggagt aggtggctac gtctccgaac
     1081 tcacgaccga aaagatcaag agcagcccgc atggatttga cttggtcagg gccgagccta
     1141 catgtgcgaa tgatgcccat acttgagcca cctaactttg ttttagggcg actgccctgc
     1201 tgcgtaacat cgttgctgct gcgtaacatc gttgctgctc cataacatca aacatcgacc
     1261 cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata gactgtacaa aaaaacagtc
     1321 ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct
     1381 ggaccagttg cgtgagcgca tacgctactt gcattacagt ttacgaaccg aacaggctta
     1441 tgtcaactgg gttcgtgcct tcatccgttt ccacggtgtg cgtcacccgg caaccttggg
     1501 cagcagcgaa gtcgaggcat ttctgtcctg gctggcgaac gagcgcaagg tttcggtctc
     1561 cacgcatcgt caggcattgg cggccttgct gttcttctac ggcaaggtgc tgtgcacgga
     1621 tctgcccttg cttcaggaga tcggtagacc tcggccgtcg cggcgcttgc cggtggtgct
     1681 gaccccggat gaagtggttc gcatcctcgg ttttctggaa ggcgagcatc gtttgttcgc
     1741 ccaggactct agctatagtt ctagtggttg gctacgtacc cgtagtggct atggcagggc
     1801 ttgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg ggttggggtg
     1861 gggaaaagga agaaacgcgg gcgtattggt cccaatgggg tctcggtggg gtatcgacag
     1921 agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca cccgtgcgtt
     1981 ttattctgtc tttttattgc cgtcatagcg cgggttcctt ccggtattgt ctccttccgt
     2041 gtttcagtta gcctccccca tctcccggta ccgcatgcta tgcatcagct gctagcacca
     2101 tggctcgaga tcccgggtga tcaagtcttc gtcgagtgat tgtaaataaa atgtaattta
     2161 cagtatagta ttttaattaa tatacaaatg atttgataat aattcttatt taactataat
     2221 atattgtgtt gggttgaatt aaaggtccgt atactccgga atattaatag atcatggaga
     2281 taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt
     2341 tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg
     2401 atcccggtcc gaagcgcgcg gaattcaaag gcctacgtcg acgagctcac tagtcgcggc
     2461 cgctttcgaa tctagagcct gcagtctcga caagcttgtc gagaagtact agaggatcat
     2521 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2581 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt ttttagtaga gacggggttt
     2641 caccatgttg gccagactgg tcttgaactc ctgacctcag gcaatctgcc cgtcttggcc
     2701 tcccaaagtg ctgggattac cggtgtgaga caccgcgccc agctgagatg ataattttta
     2761 tcttcattac ttggacaaat cattttccat ttatgggcct ctgcttttct catctgtaaa
     2821 aattgtgaat taatattgaa gaaaactgtg aatttactga atgtattatt tactgaattt
     2881 aatggttgaa tctaatggtt gaacctatta tacagttttt ttgttttgct tattttctta
     2941 tttccttggt gtttgtgtag ctgagaggcc agtacagaaa acattatttg agttacagtt
     3001 tcttttaaaa atattaattg tattctccca ttacaggtat taagaaatgg actacaaaga
     3061 ccatgacggt gattataaag atcatgatat cgattacaag gatgacgatg acaagataac
     3121 ttcgtataat gtatgctata cgaagttata agcttcacac aaaaaaccaa cacacagatg
     3181 taatgaaaat aaagatattt tatttcaggc accgggcttg cgggtcatgc accaggtgcg
     3241 cggtccttcg ggcacctcga cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg
     3301 gaggttgcgg ggcgcggagg tctccaggaa ggcgggcacc ccggcgcgct cggccgcctc
     3361 cactccgggg agcacgacgg cgctgcccag acccttgccc tggtggtcgg gcgagacgcc
     3421 gacggtggcc aggaaccacg cgggctcctt gggccggtgc ggcgccagga ggccttccat
     3481 ctgttgctgc gcggccagcc gggaaccgct caactcggcc atgcgcgggc cgatctcggc
     3541 gaacaccgcc cccgcttcga cgctctccgg cgtggtccag accgccaccg cggcgccgtc
     3601 gtccgcgacc cacaccttgc cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag
     3661 ctcggtgacc cgctcgatgt ggcggtccgg atcgacggtg tggcgcgtgg cggggtagtc
     3721 ggcgaacgcg gcggcgaggg tgcgtacggc cctggggacg tcgtcgcggg tggcgaggcg
     3781 caccgtgggc ttgtactcgg tcatgagctt tttgcaaaag cctaggcctc caaaaaagcc
     3841 tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
     3901 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     3961 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     4021 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     4081 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacaccgga tccataactt
     4141 cgtataatgt atgctatacg aagttatccg gtaccatggc agaaatcggt actggctttc
     4201 cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc
     4261 cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc
     4321 gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac ctgatcggta
     4381 tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc cgcttcatgg
     4441 atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac gactggggct
     4501 ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt attgcattta
     4561 tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc cgcgagacct
     4621 tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcag aacgttttta
     4681 tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt
     4741 accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc
     4801 tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc
     4861 tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac
     4921 cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc
     4981 cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct
     5041 ggctgtcgac gctcgagatt tccggcgagc caaccactga ggatctgtac tttcagagcg
     5101 ataacgcgat cgcttccggt accgagagaa aaataagcag aatacacctt gtttctgaac
     5161 ccagtataac tcattttcta caagtatctt gggagaaaac actggaatct ggttttgtta
     5221 ttacacttac tgatggtcat tcagcatgga ctgggacagg taatactaaa aacaaagttt
     5281 ttataagtaa aatttaagtg tgctattctt cagtcctcag ggatactttt ccattagata
     5341 ttgggtaaaa ctgatattaa ttttacttgc tattgctgtc aaatttatac acaggtttgt
     5401 aatatttaat gccactttct atatttaata gtaatagaaa aacaaacgtg tcctagaaaa
     5461 ttccggttac catttgaatc ttagtaactt ggatttgttc agaggtttaa aatgacctct
     5521 ttatagaaaa tataaatagt actttctaaa tacatctttt ctcaggaccc cagaacttgt
     5581 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5641 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5701 tctggatctg atcactgctt gagcctagga gatccgaacc agataagtga aatctagttc
     5761 caaactattt tgtcattttt aattttcgta ttagcttacg acgctacacc cagttcccat
     5821 ctattttgtc actcttccct aaataatcct taaaaactcc atttccaccc ctcccagttc
     5881 ccaactattt tgtccgccca cagcggggca tttttcttcc tgttatgttt ttaatcaaac
     5941 atcctgccaa ctccatgtga caaaccgtca tcttcggcta ctttttctct gtcacagaat
     6001 gaaaattttt ctgtcatctc ttcgttatta atgtttgtaa ttgactgaat atcaacgctt
     6061 atttgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     6121 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     6181 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     6241 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     6301 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     6361 ttggagtcca cgttcttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     6421 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     6481 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     6541 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     6601 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     6661 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     6721 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     6781 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6841 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6901 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6961 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     7021 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     7081 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     7141 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     7201 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     7261 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     7321 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     7381 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     7441 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     7501 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     7561 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     7621 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     7681 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7741 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7801 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7861 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7921 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7981 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     8041 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     8101 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     8161 aacaggagag cgcacgaggg agcttccagg
//
