LOCUS       sequence-197296-        5542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PITCh dTAG donor vector for BSD-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 5542)
  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 5542)
  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-BSD-dTAG (BRD4)
FEATURES             Location/Qualifiers
     source          1..5542
                     /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..1983
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1993..2049
                     /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             2050..2076
                     /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             2050..2076
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2080..2106
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2116..2436
                     /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(2557..2574)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2563..2674
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2792..2812)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2797..3126
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2977..3112
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3039..3058
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3175..4200
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3175..4200
                     /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    4330..4451
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4367..4386)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4421..4440
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4504..5295,5296..5364))
                     /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     5127..5146
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5365..5456)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5498..5517
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5523..5542
                     /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 gccaagcctt tgtctcaaga agaatccacc
     1621 ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc
     1681 gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt
     1741 actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc
     1801 aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga
     1861 cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt
     1921 gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag
     1981 ggcggaagcg gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac
     2041 cctggacctt acccctacga cgtgcccgac tacgccggct atccgtatga tgtcccggac
     2101 tatgcaggaa gcggaggagt gcaggtggaa accatctccc caggagacgg gcgcaccttc
     2161 cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga tggaaagaaa
     2221 gttgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa gcaggaggtg
     2281 atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc caaactgact
     2341 atatctccag attatgccta tggtgccact gggcacccag gcatcatccc accacatgcc
     2401 actctcgtct tcgatgtgga gcttctaaaa ctggaaggtg gcggtggctc gggcggtggt
     2461 gggtcgggtg gcggcggatc tggccctggg acgagattga gaaccaaaca cgtacgcgta
     2521 cgatgctcta gaatgctgat gggctagcaa aatcagcctc gactgtgcct tctagttgcc
     2581 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2641 ctgtcctttc ctaataaaat gaggaaattg catcacaaca ctcaacccta tctcggtcta
     2701 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2761 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     2821 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2881 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2941 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3001 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3061 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3121 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgatgaaa
     3181 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtt
     3241 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     3301 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     3361 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     3421 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     3481 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     3541 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     3601 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     3661 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     3721 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     3781 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     3841 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     3901 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     3961 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     4021 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     4081 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     4141 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     4201 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4261 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4321 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4381 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4441 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcattg
     4501 tcattaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4561 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4621 ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat
     4681 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4741 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4801 caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4861 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4921 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4981 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5041 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5101 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5161 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5221 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5281 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5341 gacacggaaa tgttgaatac tcatattctt cctttttcaa tattattgaa gcatttatca
     5401 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5461 ggtcagtgtt acaaccaatt aaccaattct gaacattatc gcgagcccat ttatacctga
     5521 atatggctca taacacccct tg
//
