LOCUS       sequence-494427-        4910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid expresses the degron tag knock-in sequence at the 
            N-terminus of the endogenous BRCA1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4910)
  AUTHORS   Gracia B, Zhang XH, Montes P, Pham TC, Huang M, Chen J, Karras GI
  TITLE     HSP90 buffers deleterious genetic variations in BRCA1.
  JOURNAL   Mol Cell. 2025 Nov 19:S1097-2765(25)00867-6. doi: 
            10.1016/j.molcel.2025.10.026.
  PUBMED    41265458
REFERENCE   2  (bases 1 to 4910)
  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: "Mol Cell. 
            2025 Nov 19:S1097-2765(25)00867-6. doi: 
            10.1016/j.molcel.2025.10.026."
COMMENT     Sequence Label: 
            pKlab966-pUC19-HAL-BRCA1-BSD-P2A-2xHA-FKBP12-F36V-HAR-BRCA1
FEATURES             Location/Qualifiers
     source          1..4910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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)."
     primer_bind     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2218..2541
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    2235..2291
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2292..2309)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2292..2308)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2301..2323)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2517..2536)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             3253..3648
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             3658..3714
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3715..3741
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             3715..3741
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3745..3771
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3781..4101
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     primer_bind     4860..4882
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctt gcatgcctgc aggtcgactc tagaggatcc ccgggtaccg
     2281 agctcgaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     2341 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     2401 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     2461 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     2521 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     2581 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtcttgtgg
     2641 aagaagaggt gccaggaata tgtctgggaa ggggaggaga caggattttg tgggagggag
     2701 aacttaagaa ctggatccat ttgcgccatt gagaaagcgc aagagggaag tagaggagcg
     2761 tcagtagtaa cagatgctgc cggcagggat gtgcttgagg aggatccaga gatgagagca
     2821 ggtcactggg aaaggttagg ggcggggagg ccttgattgg tgttggtttg gtcgttgttg
     2881 attttggttt tatgcaagaa aaagaaaaca accagaaaca ttggagaaag ctaaggctac
     2941 caccacctac ccggtcagtc actcctctgt agctttctct ttcttggaga aaggaaaaga
     3001 cccaaggggt tggcagcgat atgtgaaaaa attcagaatt tatgttgtct aattacaaaa
     3061 agcaacttct agaatcttta aaaataaagg acgttgtcat tagttctttg gtttgtatca
     3121 ttctaaaacc ttccaaatct taaatttact ttattttaaa atgataaaat gaagttgtca
     3181 ttttataaac cttttaaaaa gatatatata tatgtttttc taatgtgtta aagttcattc
     3241 gtagacaatc aaatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca
     3301 acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc
     3361 tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt
     3421 gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc
     3481 gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg
     3541 cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg
     3601 gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcgg aagcggagct
     3661 actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg accttacccc
     3721 tacgacgtgc ccgactacgc cggctatccg tatgatgtcc cggactatgc aggaagcgga
     3781 ggagtgcagg tggaaaccat ctccccagga gacgggcgca ccttccccaa gcgcggccag
     3841 acctgcgtgg tgcactacac cgggatgctt gaagatggaa agaaagttga ttcctcccgg
     3901 gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa
     3961 gaaggggttg cccagatgag tgtgggtcag agagccaaac tgactatatc tccagattat
     4021 gcctatggtg ccactgggca cccaggcatc atcccaccac atgccactct cgtcttcgat
     4081 gtggagcttc taaaactgga aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc
     4141 ggatctatgg atttatctgc tcttcgcgtt gaagaagtac aaaatgtcat taatgctatg
     4201 cagaaaatct tagagtgtcc catctggtaa gtcagcacaa gagtgtatta atttgggatt
     4261 cctatgatta tctcctatgc aaatgaacag aattgacctt acatactagg gaagaaaaga
     4321 catgtctagt aagattaggc tattgtaatt gctgattttc ttaactgaag aactttaaaa
     4381 atatagaaaa tgattccttg ttctccatcc actctgcctc tcccactcct ctccttttca
     4441 acacaaatcc tgtggtccgg gaaagacagg gactctgtct tgattggttc tgcactgggg
     4501 caggaatcta gtttagatta actggcattt tggcttttct tccagctcta aaacaagctc
     4561 catcacttga aatggcaaaa taaaatcatg gatgaggccg agggcggtgg cttacgcctg
     4621 taatcccagc actttgggag gccaaggtgg taggatcacg aggtcaggag atcgagacca
     4681 tcctggccaa catggtgaaa ccccctctcc actaaaaata caaaaattag ctgggcgtag
     4741 tggcatgtgc ctgtaatccc agctactcag gaggctgagg caggagaatc acttgaacca
     4801 ggaggcagat gttgctgtga gccaatatgg caccactgaa ctccagcgac agagctaaac
     4861 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//
