LOCUS       sequence-440856-        6327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template to endogenously tag NIPBL with mEGFP-mAID Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6327)
  AUTHORS   Batty P, Langer CC, Takacs Z, Tang W, Blaukopf C, Peters JM, Gerlich
            DW
  TITLE     Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2
            phase.
  JOURNAL   EMBO J. 2023 Aug 15;42(16):e113475. doi: 10.15252/embj.2023113475. 
            Epub 2023 Jun 26.
  PUBMED    37357575
REFERENCE   2  (bases 1 to 6327)
  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: "EMBO J."; 
            date: "2023-08-15"; volume: "42(16):e113475. doi"; pages: " 
            10.15252/embj.2023113475. Epub 2023 Jun 26"
COMMENT     Sequence Label: RepairTemplate_NIPBL_mEGFP_mAID
FEATURES             Location/Qualifiers
     source          1..6327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(327..329,330..332,333..1043)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(372..393)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(633..652)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     980..1001
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1068..1271
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             1320..1337
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1347..1364
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2127..2143)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2127..2143)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2140..2162)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2151..2167
                     /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(2175..2181,2182..2199,2200..2205))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2220..2241
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2358..2375)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2529..3117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2609..2628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3288..4079,4080..4148))
                     /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     3911..3930
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(4166..4960)
                     /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(4277..4296)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4887..4906
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(5304..5732)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5414..5435)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5626..5645
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5858..5880
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5872..5889
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5873..5889
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5896..5915
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5896..5914
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tcctcttagt aaaatgcaaa aaacaatagt agctactgta ctgggttgtt gtgagaactg
       61 aattaatgtg tgcaaagcac tttgcaattg cttaacatgt aataagtggt atgtgaaaat
      121 gattattata taggttgaac aaaccaaagc agtaactttt ttttatagtg attaagcatt
      181 ttcctgatag taatatatct gacatatctc tacaaataat tgtctgtttt gtgtgttgca
      241 gtgtttggga aatgggaagt aatgacagct ggcacctgaa ctaagtactt ttataggcaa
      301 caccattcca gaaattcagg gatatcatgg tgagcaaggg agaggaactg ttcaccggag
      361 tggtgcctat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
      421 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
      481 gaaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacgga gtgcagtgtt
      541 tcagccgcta ccctgaccac atgaagcagc atgacttctt caagtctgcc atgcctgaag
      601 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
      661 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
      721 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
      781 atatcatggc cgacaagcag aagaacggaa tcaaggtgaa cttcaagatc cgccacaaca
      841 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
      901 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
      961 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1021 tcggaatgga cgagctgtac aagacgcgtg gtggatccgg aggtagcaag gagaagagtg
     1081 cttgtcctaa agatccagcc aaacctccgg ccaaggcaca agttgtggga tggccaccgg
     1141 tgagatcata ccggaagaac gtgatggttt cctgccaaaa atcaagcggt ggaccagagg
     1201 cagctgcatt cgtgaaggta tcaatggacg gagcaccata cttgaggaaa atcgatttga
     1261 ggatgtataa gtactcagat ctcgagctca agcttcgaat tctgcagtcg acgagcagtc
     1321 accatcatca ccatcatagc agcggcctgg tgccgcgcgg cagccataat ggggatatgc
     1381 cccatgtccc gataacaact ctagccggga ttgctagtct cacagaccgt aagtttggtt
     1441 aatttatcta atttaagttc tactgtgtgt taacaataat ttttacagtg actgttctaa
     1501 aaattattat aggttcttta aaacacattt acagaaatag caactgaaac tgccctttaa
     1561 agaaaaaaaa aattgatagc ctaatttatc taacttctta gtgatgttta ttttggttgt
     1621 catgttcagt aatctgttgg agcttgttgg aggtatcagc agcctatttt actctaagga
     1681 cttaagaatt ttaaagaaca ttgccagtct tttgagattt atttaatgta actcaatttt
     1741 tcagagattt attcttttgt taggcatttg taactataat tcttagtcct tgaataatga
     1801 tacaaaaagc tacctacaat tttagttact gtgtcatcct acctgaataa ataaaaggaa
     1861 aaagatatga gagggaaggg tagaaagtga gaggatggca tacttttctt tggatagcat
     1921 tatgcaagat ttgggacttc ataataaggg agctaggaga aagtatcttc aagaaatgtt
     1981 tttgtccatg ttcagtatcc tccaaataca acttttctgg cttttgtttt gttttgtgaa
     2041 tttccagttg gtatctgcag aattccagca cactggcggc cgttactagt ggatccgagc
     2101 tcggtaccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2161 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2221 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2281 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2341 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2401 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2461 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2521 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2581 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2641 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2701 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2761 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2821 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2881 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2941 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3001 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3061 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3121 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3181 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3241 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3301 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3361 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3421 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3481 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3541 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3601 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3661 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3721 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3781 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3841 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3901 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3961 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4021 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4081 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4141 atactcatac tcttcctttt tcaattcaga agaactcgtc aagaaggcga tagaaggcga
     4201 tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc
     4261 cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca
     4321 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg
     4381 gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga
     4441 gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat
     4501 cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt
     4561 cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg
     4621 atactttctc ggcaggagca aggtgggatg acaggagatc ctgccccggc acttcgccca
     4681 atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc
     4741 ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg
     4801 acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg
     4861 catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag
     4921 cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg
     4981 tctcttgatc agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc
     5041 agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt
     5101 cgcttgctgt ccataaaacc gcccagtcta gctatcgcca tgtaagccca ctgcaagcta
     5161 cctgctttct ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca
     5221 tccggggtca gcaccgtttc tgcggactgg ctttctacgt gttccgcttc ctttagcagc
     5281 ccttgcgccc tgaattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5341 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5401 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5461 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5521 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5581 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5641 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5701 cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt
     5761 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     5821 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     5881 acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct agatgcatgc
     5941 tcgagcggcc gccagtgtga tggattttgt ttggaccaaa gtaggaatgg gcttcctagg
     6001 taggaagaat gtcttaagga aatgtataaa gatgggaata cacaagggag atttgggaga
     6061 atctgattag atcaggttgg ttggagaggg tggtttatat aggataatga taaaagacac
     6121 agctagaaag gcaagttgaa gctagattat gaggttaaat gtttaacgtg cttataatgt
     6181 ggaaatgaat tgcagtgctt gtcgaggttg atcaagtatt cctttctagc aatccagcct
     6241 agcagttaag aaacaaactc tggaaccaga ttgctttggt tctaattctt gctccactag
     6301 ttactttaaa ctcactgtaa attaatt
//
