LOCUS       sequence-342048-        5743 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PITCh dTAG donor vector for Puro-P2A-2xHA-FKBP_F36V knock-in into 
            the N-terminus of the human BRD4 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5743)
  AUTHORS   Nabet B, Roberts JM, Buckley DL, Paulk J, Dastjerdi S, Yang A, 
            Leggett AL, Erb MA, Lawlor MA, Souza A, Scott TG, Vittori S, Perry 
            JA, Qi J, Winter GE, Wong KK, Gray NS, Bradner JE
  TITLE     The dTAG system for immediate and target-specific protein 
            degradation.
  JOURNAL   Nat Chem Biol. 2018 May;14(5):431-441. doi: 
            10.1038/s41589-018-0021-8. Epub 2018 Mar 26.
  PUBMED    29581585
REFERENCE   2  (bases 1 to 5743)
  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"; doi: 
            "10.1038/s41589-018-0021-8"; journalName: "Nat Chem Biol"; date: 
            "2018-05"; volume: "14"; issue: "5"; pages: "431-441"
COMMENT     Sequence Label: pCRIS-PITChv2-Puro-dTAG (BRD4)
FEATURES             Location/Qualifiers
     source          1..5743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1416..1432
                     /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)."
     CDS             1588..2184
                     /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(1588..1607)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2084..2104
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2194..2250
                     /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             2251..2277
                     /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             2251..2277
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2281..2307
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2317..2637
                     /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     complement(2758..2775)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2764..2875
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2993..3013)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2998..3327
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3178..3313
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3240..3259
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3376..4401
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3376..4401
                     /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    4531..4652
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4568..4587)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4622..4641
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4705..5496,5497..5565))
                     /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     5328..5347
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5566..5657)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5699..5718
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5724..5743
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggagagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc
     1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1501 ggtcattagt tcatagccca tatatggagt tccgcgttac atagcatcgt acgcgtacgt
     1561 gtttggagca tgtctgcgga gagcggcatg accgagtaca agcccacggt gcgcctcgcc
     1621 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     1681 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     1741 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     1801 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     1861 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     1921 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     1981 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     2041 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     2101 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     2161 tgcatgaccc gcaagcccgg tgccggaagc ggagctacta acttcagcct gctgaagcag
     2221 gctggagacg tggaggagaa ccctggacct tacccctacg acgtgcccga ctacgccggc
     2281 tatccgtatg atgtcccgga ctatgcagga agcggaggag tgcaggtgga aaccatctcc
     2341 ccaggagacg ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca ctacaccggg
     2401 atgcttgaag atggaaagaa agttgattcc tcccgggaca gaaacaagcc ctttaagttt
     2461 atgctaggca agcaggaggt gatccgaggc tgggaagaag gggttgccca gatgagtgtg
     2521 ggtcagagag ccaaactgac tatatctcca gattatgcct atggtgccac tgggcaccca
     2581 ggcatcatcc caccacatgc cactctcgtc ttcgatgtgg agcttctaaa actggaaggt
     2641 ggcggtggct cgggcggtgg tgggtcgggt ggcggcggat ctggccctgg gacgagattg
     2701 agaaccaaac acgtacgcgt acgatgctct agaatgctga tgggctagca aaatcagcct
     2761 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2821 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcacaac
     2881 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2941 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3001 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3061 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3121 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3181 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3241 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3301 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3361 tctgatcagc acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     3421 atcgaaaagt tcgacagcgt ttccgacctg atgcagctct cggagggcga agaatctcgt
     3481 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     3541 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     3601 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     3661 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     3721 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     3781 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     3841 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     3901 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     3961 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     4021 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     4081 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     4141 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     4201 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     4261 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     4321 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     4381 cgtccgaggg caaaggaata gcacgtgcta cgagatttcg attccaccgc cgccttctat
     4441 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4501 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4561 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4621 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4681 tagagcttgg cgtaatcatt gtcattacca atgcttaatc agtgaggcac ctatctcagc
     4741 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4801 acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc
     4861 ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4921 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4981 ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg
     5041 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5101 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5161 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5221 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5281 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5341 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5401 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5461 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5521 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct tcctttttca
     5581 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5641 ttagaaaaat aaacaaatag gggtcagtgt tacaaccaat taaccaattc tgaacattat
     5701 cgcgagccca tttatacctg aatatggctc ataacacccc ttg
//
