LOCUS       sequence-197291-        5537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PITCh dTAG donor vector for FKBP_F36V-2xHA-P2A-BSD 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 5537)
  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 5537)
  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-BSD (BRD4)
FEATURES             Location/Qualifiers
     source          1..5537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             58..1083
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             58..1083
                     /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    1213..1334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1250..1269)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1304..1323
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1387..2178,2179..2247))
                     /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     2010..2029
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2248..2339)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2381..2400
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2406..2425
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2511..3099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3000..3019
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3253..3270
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3368..3385
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3369..3385
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3841..3857
                     /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             4058..4378
                     /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             4388..4414
                     /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             4388..4414
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4418..4444
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4448..4504
                     /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             4505..4903
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4977..4994)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4983..5094
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5212..5232)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(5217..5537,1..9)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5397..5532
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5459..5478
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgatg
       61 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
      121 gtttccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
      181 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
      241 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
      301 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
      361 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
      421 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
      481 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
      541 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
      601 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
      661 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
      721 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
      781 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
      841 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
      901 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
      961 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
     1021 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     1081 tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     1141 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     1201 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1261 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     1321 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     1381 ttgtcattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     1441 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     1501 tggccccagc gctgcgatga taccgcgaga accacgctca ccggctccgg atttatcagc
     1561 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     1621 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     1681 gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     1741 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     1801 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     1861 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     1921 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     1981 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     2041 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     2101 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     2161 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     2221 ggcgacacgg aaatgttgaa tactcatatt cttccttttt caatattatt gaagcattta
     2281 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2341 aggggtcagt gttacaacca attaaccaat tctgaacatt atcgcgagcc catttatacc
     2401 tgaatatggc tcataacacc ccttgctcat gaccaaaatc ccttaacgtg agttacgcgc
     2461 gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2521 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2581 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2641 cagataccaa atactgttct tctagtgtag ccgtagttag cccaccactt caagaactct
     2701 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2761 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2821 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2881 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2941 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3001 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3061 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3121 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3181 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3241 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag gcgagagtag ggaactgcca
     3301 ggcatcaaac taagcagaag gcccctgacg gatggccttt ttgcgtttct acaaactctt
     3361 tctgtgttgt aaaacgacgg ccagtcttaa gctcgggccc cctgggcggt tctgataacg
     3421 agtaatcgtt aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt ttatggggag
     3481 agtttaggga aagagcattt gtcagaatat ttaagggcgc ctgtcacttt gcttgatata
     3541 tgagaattat ttaaccttat aaatgagaaa aaagcaacgc actttaaata agatacgttg
     3601 ctttttcgat tgatgaacac ctataattaa actattcatc tattatttat gattttttgt
     3661 atatacaata tttctagttt gttaaagaga attaagaaaa taaatctcga aaataataaa
     3721 gggaaaatca gtttttgata tcaaaattat acatgtcaac gataatacaa aatataatac
     3781 aaactataag atgttatcag tatttattat catttagaat aaattttgtg tcgcccttaa
     3841 ttgtgagcgg ataacaatta cgagcttcat gcacagtggc gttgacattg attattgact
     3901 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     3961 gttacatagc atcgtacgcg tacgtgtttg gattgacatg aatttccaga gtggtggcgg
     4021 tggctcgggc ggtggtgggt cgggtggcgg cggatctgga gtgcaggtgg aaaccatctc
     4081 cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg
     4141 gatgcttgaa gatggaaaga aagttgattc ctcccgggac agaaacaagc cctttaagtt
     4201 tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt
     4261 gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc
     4321 aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaagg
     4381 ctcgggctac ccctacgacg tgcccgacta cgccggctat ccgtatgatg tcccggacta
     4441 tgcaggcgct actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg
     4501 acctatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac
     4561 aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga
     4621 cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact
     4681 cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat
     4741 cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga
     4801 tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg
     4861 gattcgtgaa ttgctgccct ctggttatgt gtgggagggc tgatgatcta ttgtcaatat
     4921 ttgccaaaca cgtacgcgta cgatgctcta gaatgctgat gggctagcaa aatcagcctc
     4981 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     5041 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcacaaca
     5101 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5161 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     5221 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     5281 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     5341 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     5401 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5461 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     5521 tttttggagg cctaggc
//
