LOCUS       sequence-472385-        7169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector to introduce an AID-eGFP cassette at the mouse CTCF
            locus using BLASTICIDIN selection. Auxin-inducible degron system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7169)
  AUTHORS   Nora EP, Goloborodko A, Valton AL, Gibcus JH, Uebersohn A, Abdennur 
            N, Dekker J, Mirny LA, Bruneau BG
  TITLE     Targeted Degradation of CTCF Decouples Local Insulation of 
            Chromosome Domains from Genomic Compartmentalization.
  JOURNAL   Cell. 2017 May 18;169(5):930-944.e22. doi: 
            10.1016/j.cell.2017.05.004.
  PUBMED    28525758
REFERENCE   2  (bases 1 to 7169)
  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: 
            "10.1016/j.cell.2017.05"; journalName: "Cell"; date: "2017-05-18- 
            18"; volume: "169"; issue: "5"; pages: "930-944.e22"
COMMENT     Sequence Label: pEN244 - CTCF-AID[71-114]-eGFP-FRT-Blast-FRT
            t...
FEATURES             Location/Qualifiers
     source          1..7169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     59..81
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     73..90
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     74..90
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     97..116
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        97..115
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1123..1254
                     /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(1264..1266,1267..1980)
                     /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(1264..1266,1267..1977)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1306..1327)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1567..1586)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1914..1935
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1994..2027
                     /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        2091..2590
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2126..2149)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2523..2542
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2602..2649
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     regulatory      2674..2683
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2680..3078
                     /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(3114..3131)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3120..3344
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3399..3432
                     /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(4402..4418)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4402..4418)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(4455..4473)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4494..4510)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4494..4510)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4507..4529)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4518..4534
                     /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(4542..4548,4549..4566,4567..4572))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4587..4608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4725..4742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4896..5484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4976..4995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5655..6446,6447..6515))
                     /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     6278..6297
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6516..6620)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6646..7101)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6783..6804)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6995..7014
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
       61 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
      121 ttgggtaccg ggccccccct cgaggtcgac gaagggctga agcaacgaat agactgagac
      181 agttctgctg gaggaactca ggcccctaaa gccgggagtg ctattaccag ccctaatccc
      241 aacacttggg aggctgaggc tggcctgggc tacatggaaa gaagaaggga aggggaaacc
      301 attcaggtga aaggattgtc ccaggagtgt tccctttcta gcacagtgcc tggatgtagt
      361 gggtcttaga tcgcctcaaa gcagcctttt cttctcttta aattcagggc ttaatctata
      421 attatgctcc ctgagcgcca tgtccaaatg tccatcacag aacaggggga taactctgca
      481 tctagaaccg cccagtcatt tcacctacac acaactattt cagagcagta cagggtcaga
      541 aatgtcaact gcatcatata ctctgtgaaa tactccttgt catgtgcagt ctagggctgg
      601 attttttttt ccctgccctg cattactata catcctaaat acagaacata aaggaaaact
      661 aaaaatgtta aagtggaggc ctgtgaggta aatgtcagta tttggtatct gacaatttct
      721 agccttttgg gattttatgt gtggccactt aacgttcgca gggctgtttt gtttctgctg
      781 acttgggcat cactgctgag gctttcttgt tgctgcatcc cattcattgt cagcatcggg
      841 aacaatgcct gtgctcgctg ggggctttaa tgtacgtacc ctttgttttg ttcctgccct
      901 tctttgccag caacagccat cattcaggtc gaagatcaga atacaggtgc aattgagaac
      961 attatagttg aagtcaaaaa ggagccagat gccgagcctg cggaggggga agaagaggag
     1021 gctcaggcag ccaccacaga cgcccccaac ggagacctca cgcctgagat gatcctcagc
     1081 atgatggacc gggggggagc aggaggcgtc gacggtatcg atcctaaaga tccagccaaa
     1141 cctccggcca aggcacaagt tgtgggatgg ccaccggtga gatcataccg gaagaacgtg
     1201 atggtttcct gccaaaaatc aagcggtggc ccggaggcgg cggcgttcgt gaagaagctt
     1261 gatgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1321 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1381 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1441 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1501 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1561 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1621 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1681 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1741 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1801 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1861 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1921 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     1981 gatatcgaat tccgaagttc ctattctcta gaaagtatag gaacttcagg tctgaagagg
     2041 agtttacgtc cagccaagct agcttggctg caggtcgtcg aaattctacc gggtagggga
     2101 ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg
     2161 ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc
     2221 tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc
     2281 ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag
     2341 tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg
     2401 ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg
     2461 gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc
     2521 ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc
     2581 gggcctttcg acctgcagcc tgttgacaat taatcatcgg catagtatat cggcatagta
     2641 taatacgaca aggtgaggaa ctaaacccat atggccacca tggccaagcc tttgtctcaa
     2701 gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa
     2761 gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat
     2821 gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct
     2881 gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg
     2941 agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata
     3001 gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt
     3061 tatgtgtggg agggctaacc tgagggatca attctctaga gctcgctgat cagcctcgac
     3121 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3181 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3241 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3301 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3361 aaccagctgg ggctcgacta gagcttgcgg aacccttcga agttcctatt ctctagaaag
     3421 tataggaact tcatcagtca ggtacataat ggatcctgat gctggggcct tgctcggcac
     3481 caggactatt gggctgtgtt taaacggccc aaatcttaat ttttctcttt tttttctttg
     3541 gctttgggaa cggcataatt ttacaccatt ttaccaaaca tactgagaac gaaaacttca
     3601 aggatgatgt tagaaaaatg tgatttaact agaacttgtt tgatgttagc aaatcatgga
     3661 atgttctaag tctctgaggg tttactgtga agtgttgagg acagtgttga tgcctaacta
     3721 gttttcttag atggaaacag agacattgag ccctctctct tgatcgtaaa ccactccaga
     3781 acggccacgg gtttcccaga gttctatggt cttcccaaga gaatttttaa ttgtaaatgc
     3841 agacttggga aggacttgga cttttaacct ggttttgctt ttgcttttcc tgactccctt
     3901 tgctcagagt cagttgcaca ccagtaggat ggcatgctac gatcagtttc tgtcctgaaa
     3961 gctttgcctc tttcttggca aagtttctgg tatggtcaag cttgtaaata acctttttta
     4021 cattttaatc ttttccatta attaagaggt tgaaaagaag tacagtgtaa gaaaacccag
     4081 cattttaatt atttgcaaat taaggtgcca cagacacaga cttagtggta tgtaaatgtt
     4141 gactcggatc acaatcatgt agcagagggc actgggcctg ttgcctgcca gtcatggaca
     4201 cccatgtgaa aaatcctgat ttccagctca tggagccagc gtttgaacct tgaacaaata
     4261 aatttcattt atgattttct catcaacatt ttctttgctc tgtttgtagc tgaatgttac
     4321 atttttttaa aaatctctgt tcaagccaga agagttgatt atgagtggcc acagcaaccc
     4381 tggcaagctg ggccaaaaaa ggatccacta gttctagagc ggccgccacc gcggtggagc
     4441 tccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag
     4501 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4561 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4621 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4681 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4741 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4801 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4861 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4921 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4981 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5041 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5101 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5161 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5221 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5281 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5341 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5401 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5461 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5521 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5581 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5641 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5701 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5761 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5821 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5881 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5941 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6001 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6061 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6121 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6181 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6241 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6301 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6361 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6421 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6481 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6541 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6601 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     6661 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     6721 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     6781 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     6841 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     6901 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     6961 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     7021 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     7081 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc
     7141 gatcggtgcg ggcctcttcg ctattacgc
//
