LOCUS       sequence-177754-        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector to introduce an AID-eGFP cassette at the mouse CTCF
            locus using puromycine selection. Auxin-inducible degron system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  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 7354)
  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: pEN84 - CTCF-AID[71-114]-eGFP-FRT-Puro-FRT tar...
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1027..1158
                     /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(1168..1170,1171..1884)
                     /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(1168..1170,1171..1881)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1210..1231)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1471..1490)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1818..1839
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1898..1931
                     /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        1995..2494
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2030..2053)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2427..2446
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2506..2553
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2572..3171
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2572..2591)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3068..3088
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3203..3220)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3209..3433
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3488..3521
                     /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(4491..4507)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4491..4507)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(4544..4562)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4583..4599)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4583..4599)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4596..4618)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4607..4623
                     /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(4631..4637,4638..4655,4656..4661))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4676..4697
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4814..4831)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4985..5573)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5065..5084)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5744..6535,6536..6604))
                     /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     6367..6386
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6605..6709)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6735..7190)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6872..6893)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7084..7103
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7317..7339
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7331..7348
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7332..7348
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggt cgacgaaggg
       61 ctgaagcaac gaatagactg agacagttct gctggaggaa ctcaggcccc taaagccggg
      121 agtgctatta ccagccctaa tcccaacact tgggaggctg aggctggcct gggctacatg
      181 gaaagaagaa gggaagggga aaccattcag gtgaaaggat tgtcccagga gtgttccctt
      241 tctagcacag tgcctggatg tagtgggtct tagatcgcct caaagcagcc ttttcttctc
      301 tttaaattca gggcttaatc tataattatg ctccctgagc gccatgtcca aatgtccatc
      361 acagaacagg gggataactc tgcatctaga accgcccagt catttcacct acacacaact
      421 atttcagagc agtacagggt cagaaatgtc aactgcatca tatactctgt gaaatactcc
      481 ttgtcatgtg cagtctaggg ctggattttt ttttccctgc cctgcattac tatacatcct
      541 aaatacagaa cataaaggaa aactaaaaat gttaaagtgg aggcctgtga ggtaaatgtc
      601 agtatttggt atctgacaat ttctagcctt ttgggatttt atgtgtggcc acttaacgtt
      661 cgcagggctg ttttgtttct gctgacttgg gcatcactgc tgaggctttc ttgttgctgc
      721 atcccattca ttgtcagcat cgggaacaat gcctgtgctc gctgggggct ttaatgtacg
      781 taccctttgt tttgttcctg cccttctttg ccagcaacag ccatcattca ggtcgaagat
      841 cagaatacag gtgcaattga gaacattata gttgaagtca aaaaggagcc agatgccgag
      901 cctgcggagg gggaagaaga ggaggctcag gcagccacca cagacgcccc caacggagac
      961 ctcacgcctg agatgatcct cagcatgatg gaccgggggg gagcaggagg cgtcgacggt
     1021 atcgatccta aagatccagc caaacctccg gccaaggcac aagttgtggg atggccaccg
     1081 gtgagatcat accggaagaa cgtgatggtt tcctgccaaa aatcaagcgg tggcccggag
     1141 gcggcggcgt tcgtgaagaa gcttgatgtg agcaagggcg aggagctgtt caccggggtg
     1201 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1261 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1321 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1381 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1441 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1501 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1561 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1621 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1681 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1741 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     1801 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1861 ggcatggacg agctgtacaa gtaagatatc gaattccgaa gttcctattc tctagaaagt
     1921 ataggaactt caggtctgaa gaggagttta cgtccagcca agctagcttg gctgcaggtc
     1981 gtcgaaattc taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt
     2041 agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac
     2101 atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac
     2161 tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca
     2221 aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa
     2281 gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca
     2341 gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg
     2401 cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc
     2461 gctgttctcc tcttcctcat ctccgggcct ttcgacctgc agcctgttga caattaatca
     2521 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catgaccgag
     2581 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     2641 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     2701 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     2761 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     2821 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     2881 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     2941 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     3001 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     3061 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     3121 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aggggatcaa
     3181 ttctctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3241 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3301 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3361 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3421 gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgactag agcttgcgga
     3481 acccttcgaa gttcctattc tctagaaagt ataggaactt catcagtcag gtacataatg
     3541 gatcctgatg ctggggcctt gctcggcacc aggactattg ggctgtgttt aaacggccca
     3601 aatcttaatt tttctctttt ttttctttgg ctttgggaac ggcataattt tacaccattt
     3661 taccaaacat actgagaacg aaaacttcaa ggatgatgtt agaaaaatgt gatttaacta
     3721 gaacttgttt gatgttagca aatcatggaa tgttctaagt ctctgagggt ttactgtgaa
     3781 gtgttgagga cagtgttgat gcctaactag ttttcttaga tggaaacaga gacattgagc
     3841 cctctctctt gatcgtaaac cactccagaa cggccacggg tttcccagag ttctatggtc
     3901 ttcccaagag aatttttaat tgtaaatgca gacttgggaa ggacttggac ttttaacctg
     3961 gttttgcttt tgcttttcct gactcccttt gctcagagtc agttgcacac cagtaggatg
     4021 gcatgctacg atcagtttct gtcctgaaag ctttgcctct ttcttggcaa agtttctggt
     4081 atggtcaagc ttgtaaataa ccttttttac attttaatct tttccattaa ttaagaggtt
     4141 gaaaagaagt acagtgtaag aaaacccagc attttaatta tttgcaaatt aaggtgccac
     4201 agacacagac ttagtggtat gtaaatgttg actcggatca caatcatgta gcagagggca
     4261 ctgggcctgt tgcctgccag tcatggacac ccatgtgaaa aatcctgatt tccagctcat
     4321 ggagccagcg tttgaacctt gaacaaataa atttcattta tgattttctc atcaacattt
     4381 tctttgctct gtttgtagct gaatgttaca tttttttaaa aatctctgtt caagccagaa
     4441 gagttgatta tgagtggcca cagcaaccct ggcaagctgg gccaaaaaag gatccactag
     4501 ttctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa
     4561 tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4621 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4681 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4741 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4801 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4861 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4921 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4981 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5041 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5101 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5161 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5221 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5281 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5341 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5401 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5461 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5521 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5581 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5641 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5701 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5761 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5821 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5881 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5941 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6001 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6061 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6121 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6181 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6241 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6301 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6361 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6421 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6481 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6541 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6601 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6661 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6721 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     6781 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     6841 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     6901 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     6961 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     7021 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     7081 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     7141 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     7201 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     7261 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     7321 gtcacgacgt tgtaaaacga cggccagtga attg
//
