LOCUS       sequence-310171-        7068 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector to introduce an AID-eGFP cassette at the mouse 
            RAD21 (SCC1) locus using BLASTICIDIN selection. Auxin-inducible 
            degron system. Designed to be used with sgRNA CCACGGTTCCATATTATCTG .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7068)
  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 7068)
  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: pEN527 - Rad21-AID[71-114]-eGFP-FRT-Blast-FRT
            ...
FEATURES             Location/Qualifiers
     source          1..7068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1613..1744
                     /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(1754..1756,1757..2470)
                     /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(1754..1756,1757..2467)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1796..1817)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2057..2076)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2404..2425
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2484..2517
                     /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        2581..3080
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2616..2639)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3013..3032
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3092..3139
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     regulatory      3164..3173
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3170..3568
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(3604..3621)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3610..3834
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3889..3922
                     /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(3942..3958)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3942..3958)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(4880..4898)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4919..4935)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4919..4935)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4932..4954)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4943..4959
                     /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(4967..4973,4974..4991,4992..4997))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5012..5033
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5150..5167)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5321..5909)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5401..5420)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6080..6871,6872..6940))
                     /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     6703..6722
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6941..7045)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacctcagc
      661 agatgcttca tggtcttcag gtatgccagc acagtccaca aagagtcctt tcccagaaag
      721 caagtcctgt taaaccccga agcatgcctt ggtgggaatt tctccagggc tgcattgttt
      781 taaagcatct tagttacata catgaagtgt caagccaggg gtaaacctag agcatttagt
      841 ttatgtagcc atgattatcc taaaaaacct gatgcatggt gctgtgatgg ggagcgcagc
      901 tgcgtactgt cttgtttgat tagcaaaagc catgtgctaa gtcatagagt agcagaagtc
      961 agcaacctca gctagggttc cagatacagg tgaggctgga gcacccgtga cagttctccc
     1021 aagttccttc ctttctgcac atggggatgg atgatactta gggttggaag gttatcaggg
     1081 gccggtttga ttttggtttt gttttcactt taaaatctgc tgagtgtttg tttttgctaa
     1141 ctcacatctc tgttgtggcg ccttcactta cttcttgaac tctgtatgcc ttaagataat
     1201 atgctttaga taataaggcc tttaccttag tgactagcat gggaaccact tgcgtaacta
     1261 cacgaatgtc cagtcccagc tccagccaaa cggagctcac tttgaccagt gccaaaattg
     1321 catcttctgg ttactacttt gtgcgtgagt tacttgaaat catctgcttt gttttgttct
     1381 atttcagcga gctcttgcta aaactggagc agagtctatc agtttgcttg agctgtgtcg
     1441 aaacacaaac cgaaagcagg cagcagcaaa gttctacagc tttttggttc ttaagaagca
     1501 gcaagccatc gagctcacac aggaagagcc gtacagtgac atcattgcaa cccctggacc
     1561 acggttccat attatcctcg aggggggagc aggaggcgtc gacggtatcg atcctaaaga
     1621 tccagccaaa cctccggcca aggcacaagt tgtgggatgg ccaccggtga gatcataccg
     1681 gaagaacgtg atggtttcct gccaaaaatc aagcggtggc ccggaggcgg cggcgttcgt
     1741 gaagaagctt gatgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1801 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1861 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1921 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1981 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2041 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2101 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2161 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     2221 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     2281 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2341 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2401 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2461 gtacaagtaa gatatcgaat tccgaagttc ctattctcta gaaagtatag gaacttcagg
     2521 tctgaagagg agtttacgtc cagccaagct agcttggctg caggtcgtcg aaattctacc
     2581 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     2641 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     2701 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     2761 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     2821 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     2881 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
     2941 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
     3001 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
     3061 cctcatctcc gggcctttcg acctgcagcc tgttgacaat taatcatcgg catagtatat
     3121 cggcatagta taatacgaca aggtgaggaa ctaaacccat atggccacca tggccaagcc
     3181 tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc
     3241 catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac
     3301 tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac
     3361 tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag
     3421 gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat
     3481 caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct
     3541 gccctctggt tatgtgtggg agggctaacc tgagggatca attctctaga gctcgctgat
     3601 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3661 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3721 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3781 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3841 aggcggaaag aaccagctgg ggctcgacta gagcttgcgg aacccttcga agttcctatt
     3901 ctctagaaag tataggaact tcatcagtca ggtacataat ggatccacta gttctagaca
     3961 tatgggagct agatgtgttc gagctagtga taactcacta gtacatacaa attgcccccg
     4021 tgtgcagggc accaaaaccc tttaagaaag tttttagatt tctgtttgta caaaaatctt
     4081 tgccttttct ttcttctttt tccccccagt gtttctaatt ttgtcaacca tatttttaag
     4141 ggaaactgct tatttgggtt gggtttgtat tcctggagaa aacagtagcc caagaaccca
     4201 gaagactttt aacagttcag aacagatgtg tgcaatattg gtgcatgtaa gaatatggag
     4261 taacagtcaa aaggcaccat ttttaatgtt agttttccat tactatgttg aaaggaaaac
     4321 ctgcctagga aaatgcctga cactttaaga actgtggttt gagtcccttg acaggaagag
     4381 aaaaatgtct tcccatcagt gaaaccaacg gtctggttaa ccactgtagt agggatagtg
     4441 tgtgaagcat cgtgcttgtc ttcttgatca tgtttaaagt aaatccacac tgagcaagaa
     4501 tttgtgaggt tgtgctgact gatcttcttc ggctattatg ttctgaaatc attccgtgag
     4561 tcttaggacc tctgatgcta tgcaccgcag ctcagaacga tcagacgtct acagtggcgt
     4621 gactcctcag gggctgcaga tcactctcag aggtgtttgg atgtttgctt gaataatgag
     4681 atcatggaag cagacattcc cctgcctgct gaagggcaga ccaatgagag actaccccag
     4741 attgacttcc tttaagcaaa cagtgctgta aaaactaatg gcttctctga tatttattat
     4801 aaatgttagt actcatcttt ttccaaggct gcacactcct gtagcggccg ccaccgcggt
     4861 ggagctccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg taatcatggt
     4921 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4981 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5041 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5101 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5161 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5221 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5281 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5341 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5401 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5461 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5521 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5581 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5641 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5701 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5761 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5821 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5881 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5941 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6001 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6061 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6121 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6181 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6241 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6301 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6361 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6421 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6481 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6541 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6601 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6661 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6721 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6781 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6841 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6901 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6961 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7021 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
