LOCUS       sequence-495722-        7410 bp    DNA     circular SYN 18-DEC-2025
DEFINITION  This plasmid expresses the C-terminally 3x-FLAG tagged BRCT domain 
            that contains the R1699W mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  AUTHORS   Gracia B, Montes P, Gutierrez AM, Arun B, Karras GI
  TITLE     Protein-folding chaperones predict structure-function relationships 
            and cancer risk in BRCA1 mutation carriers.
  JOURNAL   Cell Rep. 2024 Feb 27;43(2):113803. doi: 
            10.1016/j.celrep.2024.113803. Epub 2024 Feb 17.
  PUBMED    38368609
REFERENCE   2  (bases 1 to 7410)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2024-02-27"; volume: "43(2):113803. doi"; pages: " 
            10.1016/j.celrep.2024.113803. Epub 2024 Feb 17"
COMMENT     Sequence Label: pKlab224-pcDNA3-BRCA1(1314-end)-R1699W-V5-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    969..993
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2647..2671)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2673..2714
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2733..2798
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2842..2859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2848..2959
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3108..3536
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3195..3214)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3405..3426
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3545..3565)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3550..3879
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3730..3865
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3792..3811
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3928..4953
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3928..4953
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5083..5204
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5120..5139)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5174..5193
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5253..5269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5253..5269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5266..5288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5277..5293
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5301..5307,5308..5325,5326..5331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5346..5367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5484..5501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5655..6243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5735..5754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6414..7205,7206..7274))
                     /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     7037..7056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7275..7379)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
      961 agatatcaac aagtttgtac aaaaaagttg gcatggatcc tttcttgatt ggttcttcca
     1021 aacaaatgag gcatcagtct gaaagccagg gagttggtct gagtgacaag gaattggttt
     1081 cagatgatga agaaagagga acgggcttgg aagaaaataa tcaagaagag caaagcatgg
     1141 attcaaactt aggtgaagca gcatctgggt gtgagagtga aacaagcgtc tctgaagact
     1201 gctcagggct atcctctcag agtgacattt taaccactca gcagagggat accatgcaac
     1261 ataacctgat aaagctccag caggaaatgg ctgaactaga agctgtgtta gaacagcatg
     1321 ggagccagcc ttctaacagc tacccttcca tcataagtga ctcctctgcc cttgaggacc
     1381 tgcgaaatcc agaacaaagc acatcagaaa aagcagtatt aacttcacag aaaagtagtg
     1441 aataccctat aagccagaat ccagaaggcc tttctgctga caagtttgag gtgtctgcag
     1501 atagttctac cagtaaaaat aaagaaccag gagtggaaag gtcatcccct tctaaatgcc
     1561 catcattaga tgataggtgg tacatgcaca gttgctctgg gagtcttcag aatagaaact
     1621 acccatctca agaggagctc attaaggttg ttgatgtgga ggagcaacag ctggaagagt
     1681 ctgggccaca cgatttgacg gaaacatctt acttgccaag gcaagatcta gagggaaccc
     1741 cttacctgga atctggaatc agcctcttct ctgatgaccc tgaatctgat ccttctgaag
     1801 acagagcccc agagtcagct cgtgttggca acataccatc ttcaacctct gcattgaaag
     1861 ttccccaatt gaaagttgca gaatctgccc agggtccagc tgctgctcat actactgata
     1921 ctgctgggta taatgcaatg gaagaaagtg tgagcaggga gaagccagaa ttgacagctt
     1981 caacagaaag ggtcaacaaa agaatgtcca tggtggtgtc tggcctgacc ccagaagaat
     2041 ttatgctcgt gtacaagttt gccagaaaac accacatcac tttaactaat ctaattactg
     2101 aagagactac tcatgttgtt atgaaaacag atgctgagtt tgtgtgtgaa tggacactga
     2161 aatattttct aggaattgcg ggaggaaaat gggtagttag ctatttctgg gtgacccagt
     2221 ctattaaaga aagaaaaatg ctgaatgagc atgattttga agtcagagga gatgtggtca
     2281 atggaagaaa ccaccaaggt ccaaagcgag caagagaatc ccaggacaga aagatcttca
     2341 gggggctaga aatctgttgc tatgggccct tcaccaacat gcccacagat caactggaat
     2401 ggatggtaca gctgtgtggt gcttctgtgg tgaaggagct ttcatcattc acccttggca
     2461 caggtgtcca cccaattgtg gttgtgcagc cagatgcctg gacagaggac aatggcttcc
     2521 atgcaattgg gcagatgtgt gaggcacctg tggtgacccg agagtgggtg ttggacagtg
     2581 tagcactcta ccagtgccag gagctggaca cctacctgat accccagatc ccccacagcc
     2641 actactaccc aactttcttg tacaaagtgg ttggtaagcc tatccctaac cctctcctcg
     2701 gtctcgattc tacaggctcg ggctcgacct cggactacaa agaccatgac ggtgattata
     2761 aagatcatga catcgactac aaggatgacg atgacaagtg ataactcgag tctagagggc
     2821 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2881 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2941 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3001 tggggcagga cagcaagggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3061 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3121 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3181 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3241 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3301 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3361 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3421 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3481 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3541 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3601 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3661 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3721 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3781 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3841 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3901 tccattttcg gatctgatca gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc
     3961 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc
     4021 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     4081 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     4141 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc
     4201 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     4261 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     4321 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     4381 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     4441 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     4501 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     4561 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     4621 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     4681 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     4741 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     4801 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     4861 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     4921 cgccccagca ctcgtccgag ggcaaaggaa tagcacgtgc tacgagattt cgattccacc
     4981 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5041 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     5101 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5161 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     5221 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5281 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5341 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5401 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5461 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5521 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5581 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5641 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5701 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5761 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5821 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5881 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5941 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6001 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6061 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6121 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6181 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6241 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6301 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6361 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6421 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6481 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6541 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6601 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6661 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6721 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6781 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6841 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6901 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6961 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7021 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7081 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7141 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7201 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7261 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7321 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7381 acatttcccc gaaaagtgcc acctgacgtc
//
