LOCUS       sequence-345088-        7099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector to introduce an AID-eGFP cassette at the mouse WAPL
            locus using Neomycin/Kanamycin selection. Auxin-inducible degron 
            system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7099)
  AUTHORS   Liu NQ, Maresca M, van den Brand T, Braccioli L, Schijns MMGA, 
            Teunissen H, Bruneau BG, Nora EP, de Wit E
  TITLE     WAPL maintains a cohesin loading cycle to preserve 
            cell-type-specific distal gene regulation.
  JOURNAL   Nat Genet. 2021 Jan;53(1):100-109. doi: 10.1038/s41588-020-00744-4. 
            Epub 2020 Dec 14.
  PUBMED    33318687
REFERENCE   2  (bases 1 to 7099)
  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/s41588-020-00744-4"; journalName: "Nat Genet"; date: 
            "2021-01"; volume: "53"; issue: "1"; pages: "100-109"
COMMENT     Sequence Label: pNQL001-WAPL-AID[71-114]-eGFP-FRT-Neo/Kan-FRT
            ...
FEATURES             Location/Qualifiers
     source          1..7099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    4..37
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        complement(862..880)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(901..917)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(901..917)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(914..936)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    925..941
                     /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(949..955,956..973,974..979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    994..1015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1132..1149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1303..1891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1383..1402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2062..2853,2854..2922))
                     /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     2685..2704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2923..3027)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3053..3508)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3190..3211)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3402..3421
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3635..3657
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3649..3666
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3650..3666
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3673..3692
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3673..3691
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4467..4598
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     CDS             join(4608..4610,4611..5324)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4608..4610,4611..5321)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4650..4671)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4911..4930)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5258..5279
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    5338..5371
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(5470..5493)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5838..5857
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5917..5964
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5983..6786
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6046..6065)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6656..6675
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6818..6835)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6824..7048
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ttcgaagttc ctattctcta gaaagtatag gaacttcatc agtcaggtac ataatggatc
       61 cgcttggtaa tactgaagct atccttagac aaagaaaaga catgaaagaa gtccttgaag
      121 atataccaag aacattcatc agtatcattc attcgtgttg gatttttatg atcacccgat
      181 ttcttcatca tgcattctgc gtttgctaaa tgacagttac tacatcaatc tgcagctatc
      241 aaaaatgagg gaaaaggttc aggctgttaa caatcccatg cagtatttaa atacacttac
      301 ttggcagagt ctatacctgc ccctgtttcc ttgctgtatt tcaggcaagg tttaaagagg
      361 ggaatttgtg ctgctgttag tgcaactgct gtgtatgttg agccactgtt gtcatgccag
      421 tcaggtgcaa aggcagctta gctactgagg tattgaatgt tctgaggaca ttctagacaa
      481 cagcttagat cctttttcag gctcatttgc ttttgctttt tgttgaatga ttccaatcgt
      541 aaataaagct tttaataatt ttgtgaattt tttggttgtt gttccctgaa ctactgtcta
      601 tatttaaaat gagatggaat ccaaagacac aagggattaa tagtatattt ttttattctt
      661 gattaggttt gggtgttgaa ctatttttca cttctgagac catgaccata ttcaatgtca
      721 taccattatg tgtcatagct ataggcacaa gaaaacaaat agcagtttga gggaatatta
      781 tattatataa gataatgtgc cctgttaaag gattatagtt ctagagcggc cgccaccgcg
      841 gtggagctcc agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg
      901 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
      961 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     1021 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1081 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1141 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1201 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1261 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1321 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1381 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1441 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1501 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1561 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1621 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1681 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1741 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1801 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     1861 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     1921 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     1981 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2041 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2101 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2161 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2221 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2281 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2341 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2401 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2461 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2521 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2581 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2641 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2701 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     2761 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     2821 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     2881 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     2941 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3001 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     3061 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3121 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     3181 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     3241 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     3301 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     3361 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3421 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3481 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
     3541 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     3601 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     3661 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacccagac aaagtcttaa
     3721 cactgtagcc caggctggca atccacatgt ttcagccatt ggaccagtgg gacctggcta
     3781 gtttatgtgt ttttcaattt aaatgtaata catcgaaggt gaaaataata atcccatagt
     3841 gttgttcttc cctgtggcaa aatagttaac ataagcagct tacagaaggt acattttggt
     3901 ttatagttct aaatttttgt gtgtgtggtt ttttttctgt ttgtttttgt ttttttgaga
     3961 cagggtttct ctgtgtagcc ctggctgtcc tggaactcac cctgtagacc aggctggcct
     4021 cgaactcaga aatccgcctg cttctgcctc tcaagtgcta ggatcaaagg cgtgcgccac
     4081 caccaccagg ctagttctaa aatttttagt catgtgtggc aagagcagcg tggaagagca
     4141 gagtggccag ctaatagaga atgcctgtgt atctgctttc tccttttatt ccagctgtgc
     4201 ttctaggctg tgggatgatg ccacccacaa acagaaaact attgtggcag aatgttatat
     4261 tactaaggca atgatttaat ttcagtgtac tctttactac gtagactttg atactttttt
     4321 ttgtttacca ttttgttaag tattaactct aatttctctc cttcccctct tagtgtgctg
     4381 ttggaacaac aggccagaag tctatctcta gagtgattga atatttggaa cattgcggag
     4441 gagcaggagg cgtcgacggt atcgatccta aagatccagc caaacctccg gccaaggcac
     4501 aagttgtggg atggccaccg gtgagatcat accggaagaa cgtgatggtt tcctgccaaa
     4561 aatcaagcgg tggcccggag gcggcggcgt tcgtgaagaa gcttgatgtg agcaagggcg
     4621 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     4681 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     4741 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     4801 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     4861 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     4921 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     4981 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     5041 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     5101 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     5161 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     5221 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     5281 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaagatatc gaattccgaa
     5341 gttcctattc tctagaaagt ataggaactt caggtctgaa gaggagttta cgtccagcca
     5401 agctagcttg gctgcaggtc gtcgaaattc taccgggtag gggaggcgct tttcccaagg
     5461 cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc
     5521 tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc
     5581 cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc
     5641 gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac
     5701 agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc
     5761 tccttcgctt tctgggctca gaggctggga aggggtgggg ggcggggcgg gcgcccgaag
     5821 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
     5881 ctcttcctca tctccgggcc tttcgacctg cagcctgttg acaattaatc atcggcatag
     5941 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatgggatc ggccattgaa
     6001 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     6061 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     6121 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     6181 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     6241 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     6301 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     6361 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     6421 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     6481 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgatgat
     6541 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     6601 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     6661 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     6721 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     6781 ttctgagggg atcaattctc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     6841 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     6901 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     6961 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     7021 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg
     7081 actagagctt gcggaaccc
//
