LOCUS       sequence-334366-        5738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PITCh dTAG donor vector for FKBP_F36V-2xHA-P2A-Puro knock-in into 
            the C-terminus of the human BRD4 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5738)
  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 5738)
  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-dTAG-Puro (BRD4)
FEATURES             Location/Qualifiers
     source          1..5738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      99..687
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     588..607
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     841..858
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     956..973
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     957..973
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1429..1445
                     /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             1646..1966
                     /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"
     CDS             1976..2002
                     /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             1976..2002
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2006..2032
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2036..2092
                     /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             2093..2692
                     /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(2093..2112)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2589..2609
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2766..2783)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2772..2883
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3001..3021)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3006..3335
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3186..3321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3248..3267
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3384..4409
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3384..4409
                     /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    4539..4660
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4576..4595)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4630..4649
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4713..5504,5505..5573))
                     /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     5336..5355
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5574..5665)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5707..5726
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     join(5732..5738,1..13)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ataacacccc ttgctcatga ccaaaatccc ttaacgtgag ttacgcgcgc gtcgttccac
       61 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
      121 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
      181 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
      241 actgttcttc tagtgtagcc gtagttagcc caccacttca agaactctgt agcaccgcct
      301 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
      361 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
      421 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
      481 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
      541 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
      601 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
      661 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
      721 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
      781 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
      841 agcgagtcag tgagcgagga agcggaaggc gagagtaggg aactgccagg catcaaacta
      901 agcagaaggc ccctgacgga tggccttttt gcgtttctac aaactctttc tgtgttgtaa
      961 aacgacggcc agtcttaagc tcgggccccc tgggcggttc tgataacgag taatcgttaa
     1021 tccgcaaata acgtaaaaac ccgcttcggc gggttttttt atggggagag tttagggaaa
     1081 gagcatttgt cagaatattt aagggcgcct gtcactttgc ttgatatatg agaattattt
     1141 aaccttataa atgagaaaaa agcaacgcac tttaaataag atacgttgct ttttcgattg
     1201 atgaacacct ataattaaac tattcatcta ttatttatga ttttttgtat atacaatatt
     1261 tctagtttgt taaagagaat taagaaaata aatctcgaaa ataataaagg gaaaatcagt
     1321 ttttgatatc aaaattatac atgtcaacga taatacaaaa tataatacaa actataagat
     1381 gttatcagta tttattatca tttagaataa attttgtgtc gcccttaatt gtgagcggat
     1441 aacaattacg agcttcatgc acagtggcgt tgacattgat tattgactag ttattaatag
     1501 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacatagcat
     1561 cgtacgcgta cgtgtttgga ttgacatgaa tttccagagt ggtggcggtg gctcgggcgg
     1621 tggtgggtcg ggtggcggcg gatctggagt gcaggtggaa accatctccc caggagacgg
     1681 gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga
     1741 tggaaagaaa gttgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa
     1801 gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc
     1861 caaactgact atatctccag attatgccta tggtgccact gggcacccag gcatcatccc
     1921 accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaaggct ctggctaccc
     1981 ctacgacgtg cccgactacg ccggctatcc gtatgatgtc ccggactatg caggcgctac
     2041 taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac ctatgaccga
     2101 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     2161 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     2221 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     2281 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     2341 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     2401 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     2461 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     2521 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     2581 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     2641 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gatgatctat
     2701 tgtcaatatt tgccaaacac gtacgcgtac gatgctctag aatgctgatg ggctagcaaa
     2761 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2821 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2881 atcacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2941 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3001 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3061 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3121 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3181 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3241 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3301 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3361 ttttcggatc tgatcagcac gtgatgaaaa agcctgaact caccgcgacg tctgtcgaga
     3421 agtttctgat cgaaaagttc gacagcgttt ccgacctgat gcagctctcg gagggcgaag
     3481 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     3541 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     3601 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     3661 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     3721 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     3781 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     3841 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     3901 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     3961 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4021 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4081 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     4141 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     4201 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     4261 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     4321 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     4381 ccagcactcg tccgagggca aaggaatagc acgtgctacg agatttcgat tccaccgccg
     4441 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4501 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4561 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4621 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4681 cctctagcta gagcttggcg taatcattgt cattaccaat gcttaatcag tgaggcacct
     4741 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4801 actacgatac gggagggctt accatctggc cccagcgctg cgatgatacc gcgagaacca
     4861 cgctcaccgg ctccggattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4921 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4981 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg
     5041 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5101 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5161 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5221 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5281 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5341 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5401 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5461 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5521 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catattcttc
     5581 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5641 gaatgtattt agaaaaataa acaaataggg gtcagtgtta caaccaatta accaattctg
     5701 aacattatcg cgagcccatt tatacctgaa tatggctc
//
