LOCUS       sequence-471046-        7701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor for insertion of a HaloTag at the 
            C-terminus of the endogenous NBS1 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7701)
  AUTHORS   Heyza JR, Mikhova M, Bahl A, Broadbent DG, Schmidt JC
  TITLE     Systematic analysis of the molecular and biophysical properties of 
            key DNA damage response factors.
  JOURNAL   Elife. 2023 Jun 21;12:e87086. doi: 10.7554/eLife.87086.
  PUBMED    37341699
REFERENCE   2  (bases 1 to 7701)
  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: "Elife."; 
            date: "2023-06-21"; volume: "12:e87086. doi"; pages: " 
            10.7554/eLife.87086"
COMMENT     Sequence Label: NBS1-Halo HRD
FEATURES             Location/Qualifiers
     source          1..7701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3524..3529,3530..3546,3547..3552))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4060..4108
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4133..4152
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(4244..4353)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        4377..4468
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     4393..4410
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             5482..5502
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             5518..6402
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             6403..6468
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    7191..7325
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7228..7247)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7282..7301
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7354..7519
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca
     3361 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa
     3421 acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
     3481 ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca
     3541 ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac
     3601 cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc
     3661 ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg
     3721 cacggatctg cccttgcttc aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt
     3781 ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt
     3841 gttcgcccag gactctagct atagttctag tggttggcta cgtacccgta gtggctatgg
     3901 cagggcttgc cgccccgacg ttggctgcga gccctgggcc ttcacccgaa cttgggggtt
     3961 ggggtgggga aaaggaagaa acgcgggcgt attggtccca atggggtctc ggtggggtat
     4021 cgacagagtg ccagccctgg gaccgaaccc cgcgtttatg aacaaacgac ccaacacccg
     4081 tgcgttttat tctgtctttt tattgccgtc atagcgcggg ttccttccgg tattgtctcc
     4141 ttccgtgttt cagttagcct cccccatctc ccggtaccgc atgctatgca tcagctgcta
     4201 gcaccatggc tcgagatccc gggtgatcaa gtcttcgtcg agtgattgta aataaaatgt
     4261 aatttacagt atagtatttt aattaatata caaatgattt gataataatt cttatttaac
     4321 tataatatat tgtgttgggt tgaattaaag gtccgtatac tccggaatat taatagatca
     4381 tggagataat taaaatgata accatctcgc aaataaataa gtattttact gttttcgtaa
     4441 cagttttgta ataaaaaaac ctataaatat tccggattat tcataccgtc ccaccatcgg
     4501 gcgcggatcc cggtccgaag cgcgcggaat tcaaaggcct acgtcgacga gctcactagt
     4561 cgcggccgct ttcgaatcta gagcctgcag tctcgacaag cttgtcgaga agtactagag
     4621 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     4681 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttggcg gattacgagg
     4741 tcaggagttc aacactagcc tggccagcat ggagaaaccc cgtctctact aaaaatacaa
     4801 aaattagcca ggcgtggtgg catgcacctg taatcccagc tactcaggag gctgaggcag
     4861 gagaatcgct tgaacccagg aggcggaggc tgcgtgagcc aagatcgcgc cactgcactc
     4921 cagagagaga ctccatctca aaaaaaaaaa aaaaaaagtg ttgacagaca atactggttt
     4981 ttcattagtc aaatgagatg ggttgttcag ccatatttgt ttaactgctg taggagaaaa
     5041 tgttgtaaag tgctaattac atttaagtag attatgctct tttgtgcaaa tgatatgctt
     5101 ttattatggg gaggaatggg gatctttgaa gccatacaga aagaagtgta aacatacctt
     5161 catttttaaa ctgttttcat taattatttt aaagctttaa aggatttttg ttattgttga
     5221 tttttttttt gtttttacca tattttcatc aaagcaacat taaagggata catgaaaata
     5281 aaaatgtcag tgcttagtat ctgatcctaa ggacaaaatt gtcattccca tcctatttgc
     5341 caaagtgtga ctacagtttg aaagttcttt acctttaaaa gaaataccat cccttatcta
     5401 aatatcattt aaccccattt ctttctttgc agatacaatc cttatttaaa aaggagaaga
     5461 ggagctctcg agccaaccac tgaggatctg tactttcaga gcgataacga tggatccgaa
     5521 atcggtactg gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac
     5581 tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc
     5641 tcctcctacg tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct
     5701 ccagacctga tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac
     5761 cacgtccgct tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc
     5821 attcacgact ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc
     5881 aaaggtattg catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa
     5941 tttgcccgcg agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc
     6001 gatcagaacg tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa
     6061 gtcgagatgg accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg
     6121 cgcttcccaa acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa
     6181 gaatacatgg actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca
     6241 ggcgttctga tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag
     6301 gctgtggaca tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc
     6361 agcgagatcg cgcgctggct gtctactctg gagatttccg gtgactacaa agaccatgac
     6421 ggtgattata aagatcatga tatcgattac aaggatgacg atgacaagta actgaggatt
     6481 ttaaaaagaa gccatggaaa aacttcctag taagcatcta cttcaggcca acaaggttat
     6541 atgaatatat agtgtataga agcgatttaa gttacaatgt tttatggcct aaatttatta
     6601 aataaaatgc acaaaacttt gattcttttg tatgtaacaa ttgtttgttc tgttttcagg
     6661 ctttgtcatt gcatcttttt ttcattttta aatgtgtttt gtttattaaa tagttaatat
     6721 agtcacagtt caaaattcta aatgtacgta aggtaaagac taaagtcacc cttccaccat
     6781 tgtcctagct acttggttcc cctcagaaaa aaattcatga tactcatttc ttatgaatct
     6841 ttccagggat ttttgagtcc tattcaaatt cctattttta aataatttcc tacacaaatg
     6901 atagcataac atatgcagtg ttctacacct tgctttttta cttagtagat taaaaattat
     6961 aggaatatca atataatgtt tttaatattt tttcttttcc attatgctgt agtcttacct
     7021 aaactctggt gatccaaaca aaatggcttc agtggtgcag atgtcaccta catgttattc
     7081 tagtactaga aactgaagac catgtggaga cttcatcaaa catgggttta gttttcacca
     7141 gaatggaaag acctgtaccc ctttttggtg gtcttactga gctgggtggg aacttgttta
     7201 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     7261 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     7321 ggatctgatc actgcttgag cctaggagat ccgaaccaga taagtgaaat ctagttccaa
     7381 actattttgt catttttaat tttcgtatta gcttacgacg ctacacccag ttcccatcta
     7441 ttttgtcact cttccctaaa taatccttaa aaactccatt tccacccctc ccagttccca
     7501 actattttgt ccgcccacag cggggcattt ttcttcctgt tatgttttta atcaaacatc
     7561 ctgccaactc catgtgacaa accgtcatct tcggctactt tttctctgtc acagaatgaa
     7621 aatttttctg tcatctcttc gttattaatg tttgtaattg actgaatatc aacgcttatt
     7681 tgcagcctga atggcgaatg g
//
