LOCUS       sequence-432393-        8153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor for insertion of a HaloTag at the 
            N-terminus of the endogenous SHLD3 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8153)
  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 8153)
  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: Halo-SHLD3 HRD
FEATURES             Location/Qualifiers
     source          1..8153
                     /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             5126..5191
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    5192..5225
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    5232..5280
                     /note="synthetic polyadenylation signal"
     CDS             complement(5281..5880)
                     /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(5364..5384)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5861..5880
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(5887..6203)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5901..6036
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    6210..6243
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             6252..7142
                     /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             7155..7175
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     polyA_signal    7643..7777
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7680..7699)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7734..7753
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7806..7971
                     /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 gttgttgata cttccaagtc
     4741 ctttatatct attgagttaa acatgttgac tactagcgtt tttaggcagg aagtgatttt
     4801 atagggcatc tagttcagat cccttatttt ggtaagagaa agaattttca ggtttgtaga
     4861 ttacattcac atttaccaat agcactttgg taaaatacca cattgggatt ggtttttgtt
     4921 ttttgttttt ccttttctcc ctgcctgttg gtaggcaggg tatccctacc atatcttatt
     4981 ctttgcaata cttctacttt aggtcctgtt tccctctgat ttggcaagag agacaatctt
     5041 gcaataataa attaacattc tagctctgag gagttcaact aagaaatttt ctcatcacta
     5101 agagtaactg gattactgca gaatggacta caaagaccat gacggtgatt ataaagatca
     5161 tgatatcgat tacaaggatg acgatgacaa gataacttcg tataatgtat gctatacgaa
     5221 gttataagct tcacacaaaa aaccaacaca cagatgtaat gaaaataaag atattttatt
     5281 tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc
     5341 ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg cggaggtctc
     5401 caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca cgacggcgct
     5461 gcccagaccc ttgccctggt ggtcgggcga gacgccgacg gtggccagga accacgcggg
     5521 ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga
     5581 accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg cttcgacgct
     5641 ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat
     5701 gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg
     5761 gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg
     5821 tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat
     5881 gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc
     5941 agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg
     6001 agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat
     6061 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
     6121 ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
     6181 ggagcctggg gactttccac accggatcca taacttcgta taatgtatgc tatacgaagt
     6241 tatccggtac catggcagaa atcggtactg gctttccatt cgacccccat tatgtggaag
     6301 tcctgggcga gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt
     6361 tcctgcacgg taacccgacc tcctcctacg tgtggcgcaa catcatcccg catgttgcac
     6421 cgacccatcg ctgcattgct ccagacctga tcggtatggg caaatccgac aaaccagacc
     6481 tgggttattt cttcgacgac cacgtccgct tcatggatgc cttcatcgaa gccctgggtc
     6541 tggaagaggt cgtcctggtc attcacgact ggggctccgc tctgggtttc cactgggcca
     6601 agcgcaatcc agagcgcgtc aaaggtattg catttatgga gttcatccgc cctatcccga
     6661 cctgggacga atggccagaa tttgcccgcg agaccttcca ggccttccgc accaccgacg
     6721 tcggccgcaa gctgatcatc gatcagaacg tttttatcga gggtacgctg ccgatgggtg
     6781 tcgtccgccc gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg
     6841 ttgaccgcga gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga
     6901 acatcgtcgc gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc
     6961 tgctgttctg gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca
     7021 aaagcctgcc taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag
     7081 acaacccgga cctgatcggc agcgagatcg cgcgctggct gtcgacgctc gagatttccg
     7141 gcgagccaac cactgaggat ctgtactttc agagcgataa cgcgatcgct tccactacag
     7201 aagtaatatt acattatcga ccatgtgaga gtgatcccac acaactgcca aaaattgcag
     7261 aaaaagcaat tcaagacttt cctactcgtc cgctatcaag atttatacct tggtttccat
     7321 atgatgggtc caagcttcca ctcagaccta aaagatcacc acctgtgatt tctgaagagg
     7381 cagctgaaga tgtgaaacag tacttaacca tttcagaaca tgatgctaag tcacacagtt
     7441 atgattgcac agtagatcta ttggagtttc aacctagctt gaaaaagcag catttaacct
     7501 ggtcacacac actgaaggaa cagactaatt ctggaaatct gggtaaacaa tcagaaaagg
     7561 gaaaacagca caagaggaga tcttggagta tttcccttcc cagcaataat tgtactaaaa
     7621 acgtttctcc tttgtctaaa aaaacttgtt tattgcagct tataatggtt acaaataaag
     7681 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     7741 gtccaaactc atcaatgtat cttatcatgt ctggatctga tcactgcttg agcctaggag
     7801 atccgaacca gataagtgaa atctagttcc aaactatttt gtcattttta attttcgtat
     7861 tagcttacga cgctacaccc agttcccatc tattttgtca ctcttcccta aataatcctt
     7921 aaaaactcca tttccacccc tcccagttcc caactatttt gtccgcccac agcggggcat
     7981 ttttcttcct gttatgtttt taatcaaaca tcctgccaac tccatgtgac aaaccgtcat
     8041 cttcggctac tttttctctg tcacagaatg aaaatttttc tgtcatctct tcgttattaa
     8101 tgtttgtaat tgactgaata tcaacgctta tttgcagcct gaatggcgaa tgg
//
