LOCUS       sequence-494401-        6654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid expresses the C terminally 3x-FLAG tagged HALO protein 
            sequences for pulldown assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6654)
  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 6654)
  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: pKlab821-pcDNA3.1-HALO-3xFLAG
FEATURES             Location/Qualifiers
     source          1..6654
                     /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"
     CDS             999..1889
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     protein_bind    complement(1891..1915)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1917..1958
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1977..2042
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2086..2103)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2092..2203
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2352..2780
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2439..2458)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2649..2670
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2789..2809)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2794..3123
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2974..3109
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3036..3055
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3172..4197
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3172..4197
                     /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    4327..4448
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4364..4383)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4418..4437
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4497..4513)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4497..4513)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4510..4532)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4521..4537
                     /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(4545..4551,4552..4569,4570..4575))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4590..4611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4728..4745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4899..5487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4979..4998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5658..6449,6450..6518))
                     /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     6281..6300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6519..6623)
                     /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 aaaaaagcag gctccaccat ggcagaaatc ggtactggct
     1021 ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg
     1081 gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt
     1141 ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg
     1201 gtatgggcaa atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca
     1261 tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg
     1321 gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat
     1381 ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga
     1441 ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat cagaacgttt
     1501 ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct gactgaagtc gagatggacc
     1561 attaccgcga gccgttcctg aatcctgttg accgcgagcc actgtggcgc ttcccaaacg
     1621 agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact
     1681 ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg caccccaggc gttctgatcc
     1741 caccggccga agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg
     1801 gcccgggtct gaatctgctg caagaagaca acccggacct gatcggcagc gagatcgcgc
     1861 gctggctgtc gacgctcgag atttccggct acccaacttt cttgtacaaa gtggttggta
     1921 agcctatccc taaccctctc ctcggtctcg attctacagg ctcgggctcg acctcggact
     1981 acaaagacca tgacggtgat tataaagatc atgacatcga ctacaaggat gacgatgaca
     2041 agtgataact cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     2101 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2161 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2221 gtcattctat tctggggggt ggggtggggc aggacagcaa ggggaagaca atagcaggca
     2281 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     2341 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2401 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2461 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2521 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2581 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2641 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2701 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2761 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     2821 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     2881 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2941 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3001 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3061 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3121 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt gatgaaaaag
     3181 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
     3241 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
     3301 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
     3361 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc
     3421 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
     3481 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
     3541 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
     3601 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     3661 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     3721 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     3781 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     3841 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     3901 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     3961 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     4021 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     4081 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     4141 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac
     4201 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4261 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4321 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4381 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4441 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4501 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4561 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4621 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4681 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4741 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4801 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4861 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4921 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4981 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5041 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5101 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5161 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5221 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5281 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5341 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5401 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5461 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5521 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5581 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5641 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5701 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5761 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5821 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5881 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5941 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6001 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6061 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6121 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6181 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6241 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6301 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6361 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6421 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6481 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6541 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6601 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
