LOCUS       sequence-323555-        6899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acute Depletion of NIPBL-EYFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6899)
  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 6899)
  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_N-Nipbl_BSD_FKBPF36V_EYFP
FEATURES             Location/Qualifiers
     source          1..6899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1114..1509
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1519..1575
                     /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             1576..1599
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1609..1929
                     /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             1936..1962
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1963..1965,1966..2676)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2005..2026)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2266..2285)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2613..2634
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(4402..4420)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4441..4457)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4441..4457)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4454..4476)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4465..4481
                     /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(4489..4495,4496..4513,4514..4519))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4534..4555
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4672..4689)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4843..5431)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4923..4942)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5602..6393,6394..6462))
                     /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     6225..6244
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6463..6567)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6635..6653
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6691..6713)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6813..6832
                     /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 ggtcgaccag actcttgtgc
      241 tgagggaaag atcctagttc cgcagattat cttctgatct gcacacacat cagtcatagc
      301 aggcacacaa caatacacgt ggacactaag tgaacaaata gtaacaaatt acaaatgggg
      361 caggggagga ggagggacat atcaagtgtg tcacaaagga atatacagca atttactgag
      421 aaaatattgg gcaataaatg tcataaattc agttatccta aggtaaaatg gctgtggaat
      481 gaaagtgttc ttgaattgta ggatttagaa gagaggagaa taaaagtctc acactgcaaa
      541 ccacaaggat ccaaaagaat ctgaagagat actcttggta ggaggggcaa gtccatttag
      601 agagtaaaaa gggaactaga caagaaattg aagccagatt attaagtaaa aattttattg
      661 tatttagagt ttagaaagag taccctgccc tagcagtata tacaccagaa gttaagaaac
      721 aagattctgg agctagattg tgtcattaga attacagctc cagtagttac tttaaactca
      781 ctgtaatttt ctcattagta aaatgcagaa aagagtaata ctctgctgta cctgggttgt
      841 tgtgagacct aaatcttaat gtatacaaag cattttgcag tgtttaatgt atggcacatg
      901 cttgttaaac acattgaaca aatagaagct aagagttttg tttctaaatg aataagtatc
      961 tctctgctac tcatatattt gacaaagttc tacaaataat tatatggttt tctttgtgtg
     1021 tgttgcagag tttgggaaat ggaaagtaat gacagctggc acctgaacta aatactttca
     1081 tagacaacac cattccagaa cttcagggtc gacatggcca agcctttgtc tcaagaagaa
     1141 tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac
     1201 agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat
     1261 cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca
     1321 gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc
     1381 tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag
     1441 gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg
     1501 tgggagggcg gttccggagc cacgaacttc tctctgttaa agcaagcagg agacgtggaa
     1561 gaaaaccccg gtcccgatta caaggatgac gatgacaagg gttccggcgg cgtgcaggtg
     1621 gagactatct ccccaggaga cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg
     1681 cactacaccg ggatgcttga agatggaaag aaagttgatt cctcccggga cagaaacaag
     1741 ccctttaagt ttatgctagg caagcaggag gtgatccgag gctgggaaga aggggttgcc
     1801 cagatgagtg tgggtcagag agccaaactg actatatctc cagattatgc ctatggtgcc
     1861 actgggcacc caggcatcat cccaccacat gccactctcg tcttcgatgt ggagcttcta
     1921 aaactggaag tcgattatcc gtacgacgta ccagactacg cagtgagcaa gggcgaggag
     1981 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2041 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     2101 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
     2161 ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2221 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2281 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2341 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2401 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2461 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2521 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc
     2581 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2641 gccgggatca ctctcggcat ggacgagctg tacaagggtg gaggtggatc tggtggcggc
     2701 ggttcaggta ccaatgggga tatgccccat gtccccataa ctacgcttgc ggggattgct
     2761 agtcttacgg accgtaagtt tgcataattt tcctaagttc tttactttgc taatactcat
     2821 ttttagagac aaaaaattac ctttaatgtc acatataaca gaaaatatgt atttgattag
     2881 tgtgggggga gtagcaaaat taatgtttcc acctaataca taaatttaat ttcttcatcc
     2941 ttctgtgaac tcctaaagaa atgatagtgt gataattttt ataaagacct taaaaatcaa
     3001 taaggacttt agaaaaatga cacaaagctt taagagatgt taaaacttaa ataaacatgg
     3061 gttaataaac aaaactatgg tctttttttt aagaaagcgt ttacaaaaac agtacctgaa
     3121 agtgtcctcc aagaatgaga aattgatcgc ccacattacc taaactgtag tggtcctgat
     3181 ttttttaata tcaggttgaa ttgttagtta gaatttgtta acaggtatga gatatgtcat
     3241 cactatgaga gcccacttta ctctgaacac taaggtatta aaaattattg tcaatactta
     3301 gaaatttagt taatagaagt tttaaggttc atattttggt agacacataa ctattctttt
     3361 ccttgggtaa tggcacagag tctgcccaag tgtttgtccc tatgtcatcc tccttttagt
     3421 ataggaatag agataaagga aaggccagaa aagaaaaaac aaaatgaaaa cataaatacc
     3481 tttttcttat agcagtatgt aagatatgga actttgcaat aatctatgaa aaaataactt
     3541 ccagaactgt tttcaactgt gctcagaatc cctccctggg ttttgttttt gtgtattttc
     3601 agtcgagtgt aagacacttg tatcactaga aagtaggtgg ttggattatc ctaaatctgc
     3661 agggaaactt tgaaacatgt ttgtttttgt tactcgtttt aaggcaaaaa cacaacctgt
     3721 atggatggaa aattatatat gctgctagac ttccctttat cttttctgcc ttttatctct
     3781 gatatacctc acatttttga tgcttatctt cctggtaaac tctgtgaagt atggtaatta
     3841 tactatgggg tcgactagaa attaacccca aaacatgata cctatatttt agttgggttt
     3901 cttaaccttt tggcaagtaa acaattccta ttaagtctac ttacattaaa tagtgggatt
     3961 gaagccacaa aatactcatt tgtttgtttg tttgtttgtt tgctgcttag agtagaatgg
     4021 gagaaaaaag agaactattt aggaatttat gacaagaatt ttgtattgtt cttcaggaat
     4081 gttgtactct gagtattcca ctactgtgag ctatttgttt ctatattctt aagcttttaa
     4141 aattgtattg catatcagta taatgtgagt caggtattgt ttttgactta tatgtctaag
     4201 agtgctactg atcttataga taagctactt tctcacctta tgtgtcaaag tcatgataat
     4261 aataataaag ttttatttag ttaattaaaa caatttttaa gcatgaatat aaatcagcta
     4321 ggtaccctcg aggtcgatcg acggtatcga taagcttgat ctagagcggc cgccaccgcg
     4381 gtggagctcc agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg
     4441 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4501 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4561 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4621 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4681 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4741 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4801 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4861 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4921 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4981 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5041 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5101 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5161 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5221 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5281 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5341 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5401 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5461 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5521 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5581 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5641 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5701 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5761 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5821 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5881 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5941 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6001 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6061 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6121 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6181 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6241 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6301 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6361 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6421 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6481 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6541 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6601 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6661 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     6721 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     6781 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     6841 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggc
//
