LOCUS       sequence-323478-        7322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acute Depletion NIPBL-mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7322)
  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 7322)
  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_Neo_FKBPF36V_mScarlet
FEATURES             Location/Qualifiers
     source          1..7322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1114..1905
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1168..1187)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1778..1797
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             1915..1971
                     /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             1972..1998
                     /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             1972..1998
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2002..2028
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2038..2358
                     /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             2389..3084
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2522..2540)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2790..2809
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(4825..4843)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4864..4880)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4864..4880)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4877..4899)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4888..4904
                     /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(4912..4918,4919..4936,4937..4942))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4957..4978
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5095..5112)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5266..5854)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5346..5365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6025..6816,6817..6885))
                     /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     6648..6667
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6886..6990)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7058..7076
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7114..7136)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7236..7255
                     /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 gacatgattg aacaagatgg attgcacgca
     1141 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     1201 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     1261 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     1321 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     1381 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     1441 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     1501 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     1561 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     1621 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     1681 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     1741 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     1801 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     1861 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttcggttc cggagctact
     1921 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc ttacccctac
     1981 gacgtgcccg actacgccgg ctacccatac gatgttccag attacgctgg aagcggagga
     2041 gtgcaggtgg aaaccatctc cccaggagac gggcgcacct tccccaagcg cggccagacc
     2101 tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga aagttgattc ctcccgggac
     2161 agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg ctgggaagaa
     2221 ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc agattatgcc
     2281 tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt cttcgatgtg
     2341 gagcttctaa aactggaagg tggaggcggg agtggaggtg gaggatcgat ggtgagcaag
     2401 ggcgaggcag tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac
     2461 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     2521 gccaagctga aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct
     2581 cagttcatgt acggctccag ggccttcatc aagcaccccg ccgacatccc cgactactat
     2641 aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     2701 gccgtgaccg tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag
     2761 ctccgcggca ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg
     2821 gaagcgtcca ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg
     2881 gccctgcgcc tgaaggacgg cggccgctac ctggcggact tcaagaccac ctacaaggcc
     2941 aagaagcccg tgcagatgcc cggcgcctac aacgtggacc gcaagttgga catcacctcc
     3001 cacaacgagg actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc
     3061 ggcggcatgg acgagctgta caagggtggc ggtggctcgg gcggtggtgg gtcgggtggc
     3121 ggcggatctg gtaccaatgg ggatatgccc catgtcccca taactacgct tgcggggatt
     3181 gctagtctta cggaccgtaa gtttgcataa ttttcctaag ttctttactt tgctaatact
     3241 catttttaga gacaaaaaat tacctttaat gtcacatata acagaaaata tgtatttgat
     3301 tagtgtgggg ggagtagcaa aattaatgtt tccacctaat acataaattt aatttcttca
     3361 tccttctgtg aactcctaaa gaaatgatag tgtgataatt tttataaaga ccttaaaaat
     3421 caataaggac tttagaaaaa tgacacaaag ctttaagaga tgttaaaact taaataaaca
     3481 tgggttaata aacaaaacta tggtcttttt tttaagaaag cgtttacaaa aacagtacct
     3541 gaaagtgtcc tccaagaatg agaaattgat cgcccacatt acctaaactg tagtggtcct
     3601 gattttttta atatcaggtt gaattgttag ttagaatttg ttaacaggta tgagatatgt
     3661 catcactatg agagcccact ttactctgaa cactaaggta ttaaaaatta ttgtcaatac
     3721 ttagaaattt agttaataga agttttaagg ttcatatttt ggtagacaca taactattct
     3781 tttccttggg taatggcaca gagtctgccc aagtgtttgt ccctatgtca tcctcctttt
     3841 agtataggaa tagagataaa ggaaaggcca gaaaagaaaa aacaaaatga aaacataaat
     3901 acctttttct tatagcagta tgtaagatat ggaactttgc aataatctat gaaaaaataa
     3961 cttccagaac tgttttcaac tgtgctcaga atccctccct gggttttgtt tttgtgtatt
     4021 ttcagtcgag tgtaagacac ttgtatcact agaaagtagg tggttggatt atcctaaatc
     4081 tgcagggaaa ctttgaaaca tgtttgtttt tgttactcgt tttaaggcaa aaacacaacc
     4141 tgtatggatg gaaaattata tatgctgcta gacttccctt tatcttttct gccttttatc
     4201 tctgatatac ctcacatttt tgatgcttat cttcctggta aactctgtga agtatggtaa
     4261 ttatactatg gggtcgacta gaaattaacc ccaaaacatg atacctatat tttagttggg
     4321 tttcttaacc ttttggcaag taaacaattc ctattaagtc tacttacatt aaatagtggg
     4381 attgaagcca caaaatactc atttgtttgt ttgtttgttt gtttgctgct tagagtagaa
     4441 tgggagaaaa aagagaacta tttaggaatt tatgacaaga attttgtatt gttcttcagg
     4501 aatgttgtac tctgagtatt ccactactgt gagctatttg tttctatatt cttaagcttt
     4561 taaaattgta ttgcatatca gtataatgtg agtcaggtat tgtttttgac ttatatgtct
     4621 aagagtgcta ctgatcttat agataagcta ctttctcacc ttatgtgtca aagtcatgat
     4681 aataataata aagttttatt tagttaatta aaacaatttt taagcatgaa tataaatcag
     4741 ctaggtaccc tcgaggtcga tcgacggtat cgataagctt gatctagagc ggccgccacc
     4801 gcggtggagc tccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc
     4861 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4921 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4981 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5041 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5101 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5161 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5221 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5281 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5341 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5401 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5461 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5521 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5581 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5641 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5701 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5761 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5821 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5881 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5941 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6001 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6061 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6121 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6181 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6241 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6301 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6361 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6421 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6481 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6541 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6601 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6661 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6721 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6781 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6841 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6901 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6961 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7021 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7081 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     7141 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     7201 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     7261 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     7321 gc
//
