LOCUS       sequence-432324-        8409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor for insertion of a HaloTag at the 
            C-terminus of the endogenous RIF1 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8409)
  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 8409)
  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: Rif1-Halo HRD
FEATURES             Location/Qualifiers
     source          1..8409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      102..556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(189..208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     398..419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        582..686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(687..755,756..1547)
                     /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(905..924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1718..2306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2207..2226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2460..2477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2611..2835
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2902..3435)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3624..3629,3630..3646,3647..3652))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4160..4208
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4233..4252
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(4344..4453)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        4477..4568
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     4493..4510
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             5297..5317
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             5333..6217
                     /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             6218..6283
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    6316..6540
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        6562..6891
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6742..6877
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6901..7500
                     /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(6901..6920)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7397..7417
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7501..7549
                     /note="synthetic polyadenylation signal"
     polyA_signal    7999..8133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8036..8055)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8090..8109
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  8162..8327
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga
       61 ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
      121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      301 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      361 ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga
      421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
      481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
      541 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      601 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
      661 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
      721 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
      781 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
      841 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
      901 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
      961 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1021 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1081 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1141 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1201 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     1261 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1321 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1381 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     1441 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1501 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     1561 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     1681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1861 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2101 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2161 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2221 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2281 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2341 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2401 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2461 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2521 tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg
     2581 agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa
     2641 caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga
     2701 aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact
     2761 cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg
     2821 gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc
     2881 gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac
     2941 ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc
     3001 ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag
     3061 cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat
     3121 ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc
     3181 ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta
     3241 atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag
     3301 atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat
     3361 gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt
     3421 gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg
     3481 cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga
     3541 aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg
     3601 agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc
     3661 gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg
     3721 aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg
     3781 cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc
     3841 aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag
     3901 tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct
     3961 atagttctag tggttggcta cgtacccgta gtggctatgg cagggcttgc cgccccgacg
     4021 ttggctgcga gccctgggcc ttcacccgaa cttgggggtt ggggtgggga aaaggaagaa
     4081 acgcgggcgt attggtccca atggggtctc ggtggggtat cgacagagtg ccagccctgg
     4141 gaccgaaccc cgcgtttatg aacaaacgac ccaacacccg tgcgttttat tctgtctttt
     4201 tattgccgtc atagcgcggg ttccttccgg tattgtctcc ttccgtgttt cagttagcct
     4261 cccccatctc ccggtaccgc atgctatgca tcagctgcta gcaccatggc tcgagatccc
     4321 gggtgatcaa gtcttcgtcg agtgattgta aataaaatgt aatttacagt atagtatttt
     4381 aattaatata caaatgattt gataataatt cttatttaac tataatatat tgtgttgggt
     4441 tgaattaaag gtccgtatac tccggaatat taatagatca tggagataat taaaatgata
     4501 accatctcgc aaataaataa gtattttact gttttcgtaa cagttttgta ataaaaaaac
     4561 ctataaatat tccggattat tcataccgtc ccaccatcgg gcgcggatcc cggtccgaag
     4621 cgcgcggaat tcaaaggcct acgtcgacga gctcactagt cgcggccgct ttcgaatcta
     4681 gagcctgcag tctcgacaag cttgtcgaga agtactagag gatcataatc agccatacca
     4741 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     4801 ataaaatgaa tgcaattgtt gttgttgagc tgagattacg ccactgcact ccagcctggg
     4861 caatagaatg aggctccatc tcataataat aataagcagc agcggcagct ctggtagatt
     4921 tttttgtgtg cttgtcctca tgaagactta agcctgctct ctcaatttga aacctaggca
     4981 cttggactaa cttaaaagat catgtaaaaa atttaatttt tgtctggtat agatttaatt
     5041 gtggtttttt ttttctcttt tagatataat tgatcctgtt gctttagaaa ttccattatc
     5101 caaaaacctt ctggcacaga ttagtgctct tgctcttcag ctggattcag aagatcttca
     5161 taattattca ggaagccaac tatttgaaat gcacgagaaa ctaagttgta tggcaaactc
     5221 tgtaataaaa aatctacagt cacgttggag atcaccatcc catgaaaatt ctattggagc
     5281 tctcgagcca accactgagg atctgtactt tcagagcgat aacgatggat ccgaaatcgg
     5341 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     5401 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     5461 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     5521 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     5581 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     5641 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     5701 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     5761 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     5821 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     5881 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     5941 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     6001 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     6061 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     6121 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     6181 gatcgcgcgc tggctgtcta ctctggagat ttccggtgac tacaaagacc atgacggtga
     6241 ttataaagat catgatatcg attacaagga tgacgatgac aagtaactcg agtctagacg
     6301 tttaaaccct gcaggctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     6361 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     6421 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     6481 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     6541 gtcgacagta ctaagcttta cgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     6601 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     6661 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     6721 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     6781 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     6841 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttacc
     6901 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     6961 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     7021 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     7081 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     7141 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     7201 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     7261 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     7321 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     7381 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     7441 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     7501 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtgt attttcagag
     7561 aaaattgaag gtttttttaa acatcactgg atttcttgat tgaggaaaca agttctgaaa
     7621 taatagcaca atttcaaaga agagactctt tgcaaagttg ataacatttc aaaccctgaa
     7681 ggacagtgac ttattatgta agttcaattt tgtaagttca ttatgtaaga tccttttttt
     7741 tttcataata tgtattcttg gctgctatgc gtggtttttc aggaaattta attatcttac
     7801 tgagatgtga aagcaaaact agtaacagaa cttacatttt atttcatgct ttcttaaacc
     7861 cgtgcatatt ctggtgaaac atgtaaaata ctttaagtaa aattgaacat ttttatttga
     7921 atttttgctg aactgataaa ggtgtttata tttttgtttg ttggtttgtt taattcatgt
     7981 ttgttgggac tgaggtttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     8041 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     8101 aaactcatca atgtatctta tcatgtctgg atctgatcac tgcttgagcc taggagatcc
     8161 gaaccagata agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc
     8221 ttacgacgct acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa
     8281 actccatttc cacccctccc agttcccaac tattttgtcc gcccacagcg gggcattttt
     8341 cttcctgtta tgtttttaat caaacatcct gccaactcca tgtgacaaac cgtcatcttc
     8401 ggctacttt
//
