LOCUS       sequence-323554-        6693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acute Depletion of CTCF-EYFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6693)
  AUTHORS   Zhu Y, Denholtz M, Lu H, Murre C
  TITLE     Calcium signaling instructs NIPBL recruitment at active enhancers 
            and promoters via distinct mechanisms to reconstruct genome 
            compartmentalization.
  JOURNAL   Genes Dev. 2021 Jan 1;35(1-2):65-81. doi: 10.1101/gad.343475.120. 
            Epub 2020 Dec 17.
  PUBMED    33334824
REFERENCE   2  (bases 1 to 6693)
  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: "Genes 
            Dev."; date: "2021-01-1"; volume: "35(1-2):65-81. doi"; pages: " 
            10.1101/gad.343475.120. Epub 2020 Dec 17"
COMMENT     Sequence Label: pFlox c-CTCF FKBPF36V_EYFP_p2aBsd
FEATURES             Location/Qualifiers
     source          1..6693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1623..1646
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1656..1976
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1983..2009
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2010..2012,2013..2723)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2052..2073)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2313..2332)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2660..2681
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2733..2789
                     /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             2790..3188
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(4196..4214)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4235..4251)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4235..4251)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4248..4270)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4259..4275
                     /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(4283..4289,4290..4307,4308..4313))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4328..4349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4466..4483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4637..5225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4717..4736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5396..6187,6188..6256))
                     /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     6019..6038
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6257..6361)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6429..6447
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6485..6507)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6607..6626
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      121 ggttttccca gtcacgacgt tgtaaaacga cggccgcggc ggccgcacta gtggccgcgg
      181 cggccgcact agtggccgcg gcggccgcag cttgcctgca ggtcgactgg aagaaccaac
      241 cagcccaaac agaaccagcg taagttgcct gccctggctc agtgagcatc tcatgtgagg
      301 aagcagactg tgtttgtgaa gatgggcact gagctttgag aacaagtgaa ggtcaccaca
      361 caggcacaga tgctcaggaa gccctgtttt gtttgtttgg tttggtttgg ttttttggtt
      421 ttttggtttt ttaaggaagt ttttttgttt ttgttttgtt ttttgatttt ggttttttgt
      481 ttttttactg agaaacatct gaggcagtct ctaaaagcct gttacaaatg aaaaagtcta
      541 gaggagaagg gtactaatgt cctgcaagat gggtgtgctg ttattttggg agcgattata
      601 aaacaaccaa ggtcacagtc acatggcaga gctgacatgg ttataagtag aaaagagcca
      661 ggctttcagg gaatgaaggg ctgaagcaac gaatagactg agacagttct gctggaggaa
      721 ctcaggcccc taaagccggg agtgctatta ccagccctaa tcccaacact tgggaggctg
      781 aggctggcct gggctacatg gaaagaagaa gggaagggga aaccattcag gtgaaaggat
      841 tgtcccagga gtgttccctt tctagcacag tgcctggatg tagtgggtct tagatcgcct
      901 caaagcagcc ttttcttctc tttaaattca gggcttaatc tataattatg ctccctgagc
      961 gccatgtcca aatgtccatc acagaacagg gggataactc tgcatctaga accgcccagt
     1021 catttcacct acacacaact atttcagagc agtacagggt cagaaatgtc aactgcatca
     1081 tatactctgt gaaatactcc ttgtcatgtg cagtctaggg ctggattttt ttttccctgc
     1141 cctgcattac tatacatcct aaatacagaa cataaaggaa aactaaaaat gttaaagtgg
     1201 aggcctgtga ggtaaatgtc agtatttggt atctgacaat ttctagcctt ttgggatttt
     1261 atgtgtggcc acttaacgtt cgcagggctg ttttgtttct gctgacttgg gcatcactgc
     1321 tgaggctttc ttgttgctgc atcccattca ttgtcagcat cgggaacaat gcctgtgctc
     1381 gctgggggct ttaatgtacg taccctttgt tttgttcctg cccttctttg ccagcaacag
     1441 ccatcattca ggtcgaagat cagaatacag gtgcaattga gaacattata gttgaagtca
     1501 aaaaggagcc agatgccgag cctgcggagg gggaagaaga ggaggctcag gcagccacca
     1561 cagacgcccc caacggagac ctcacgcctg agatgatcct cagcatgatg gaccgggtcg
     1621 acgattacaa ggatgacgat gacaagggtt ccggcggcgt gcaggtggag actatctccc
     1681 caggagacgg gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga
     1741 tgcttgaaga tggaaagaaa gttgattcct cccgggacag aaacaagccc tttaagttta
     1801 tgctaggcaa gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg
     1861 gtcagagagc caaactgact atatctccag attatgccta tggtgccact gggcacccag
     1921 gcatcatccc accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaagtcg
     1981 attatccgta cgacgtacca gactacgcag tgagcaaggg cgaggagctg ttcaccgggg
     2041 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2101 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2161 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgcagtgct
     2221 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2281 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2341 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2401 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2461 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2521 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2581 gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc
     2641 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2701 tcggcatgga cgagctgtac aagggatccg gcgcaacaaa cttctctctg ctgaaacaag
     2761 ccggagatgt cgaagagaat cctggaccga tggccaagcc tttgtctcaa gaagaatcca
     2821 ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg
     2881 tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt
     2941 ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg
     3001 gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg
     3061 gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca
     3121 gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg
     3181 agggctaagg tacctgctgg ggcgttgctc ggcaccagga ctattgggct gtgtttaaac
     3241 ggcccaaatc ttaatttttc tctttttttt ctttggcttt gggaacggca taattttaca
     3301 ccattttacc aaacatactg agaacgaaaa cttcaaggat gatgttagaa aaatgtgatt
     3361 taactagaac ttgtttgatg ttagcaaatc atggaatgtt ctaagtctct gagggtttac
     3421 tgtgaagtgt tgaggacagt gttgatgcct aactagtttt cttagatgga aacagagaca
     3481 ttgagccctc tctcttgatc gtaaaccact ccagaacggc cacgggtttc ccagagttct
     3541 atggtcttcc caagagaatt tttaattgta aatgcagact tgggaaggac ttggactttt
     3601 aacctggttt tgcttttgct tttcctgact ccctttgctc agagtcagtt gcacaccagt
     3661 aggatggcat gctacgatca gtttctgtcc tgaaagcttt gcctctttct tggcaaagtt
     3721 tctggtatgg tcaagcttgt aaataacctt ttttacattt taatcttttc cattaattaa
     3781 gaggttgaaa agaagtacag tgtaagaaaa cccagcattt taattatttg caaattaagg
     3841 tgccacagac acagacttag tggtatgtaa atgttgactc ggatcacaat catgtagcag
     3901 agggcactgg gcctgttgcc tgccagtcat ggacacccat gtgaaaaatc ctgatttcca
     3961 gctcatggag ccagcgtttg aaccttgaac aaataaattt catttatgat tttctcatca
     4021 acattttctt tgctctgttt gtagctgaat gttacatttt tttaaaaatc tctgttcaag
     4081 ccagaagagt tgattatgag tggccggtac cgggcccccc ctcgaggtcg atcgacggta
     4141 tcgataagct tgatctagag cggccgccac cgcggtggag ctccagcttt tgttcccttt
     4201 agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4261 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4321 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4381 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4441 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4501 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4561 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4621 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4681 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4741 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4801 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4861 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4921 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4981 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5041 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5101 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5161 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5221 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5281 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5341 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5401 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5461 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5521 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5581 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5641 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5701 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5761 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5821 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5881 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5941 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6001 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6061 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6121 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6181 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6241 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6301 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6361 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6421 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     6481 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     6541 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     6601 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     6661 acagatgcgt aaggagaaaa taccgcatca ggc
//
