LOCUS       sequence-385205-        8095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector to introduce human CTCF cDNA tagged with eGFP and two AIDs in
            the cells. Blasticidin selection (2A-fusion). Auxin-inducible degron
            system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8095)
  AUTHORS   Valton AL, Venev SV, Mair B, Khokhar ES, Tong AHY, Usaj M, Chan K, 
            Pai AA, Moffat J, Dekker J
  TITLE     A cohesin traffic pattern genetically linked to gene regulation.
  JOURNAL   Nat Struct Mol Biol. 2022 Dec 8. doi: 10.1038/s41594-022-00890-9.
  PUBMED    36482254
REFERENCE   2  (bases 1 to 8095)
  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: "Nat Struct 
            Mol Biol. 2022 Dec 8. doi: 10.1038/s41594-022-00890-9."
COMMENT     Sequence Label: 221-pCAGGS-HA-AID-CTCFcDNA-AID-eGFP-2A-blastic...
FEATURES             Location/Qualifiers
     source          1..8095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        581..960
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        962..1238
                     /label=chicken beta-actin promoter
     intron          1239..2255
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2179..2201
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2263..2282
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2337..2363
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2379..2510
                     /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             4734..4865
                     /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(4875..4877,4878..5588)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4875..4877,4878..5588)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4917..4938)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5178..5197)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5525..5546
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5598..5651
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(6096..6113)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6102..6309
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6336..6355)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6337..6355)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6360..6376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6360..6376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6478..6497)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6489..7298
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(6556..6575)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      7445..8033
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7934..7953
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tatttaaatg gcgcgccgtc gacattgatt attgactagt
      601 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      661 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      721 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      781 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      841 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      901 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      961 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
     1021 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg
     1081 gncgcgcgcc aggsggggcg gggsggggsg rggggsgggg sggggcgagg cggagaggtg
     1141 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
     1201 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     1261 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1321 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg
     1381 tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg
     1441 gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg
     1501 ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt
     1561 gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg
     1621 ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg
     1681 gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc
     1741 acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg
     1801 gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggrgg
     1861 gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc
     1921 gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa
     1981 tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga
     2041 agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc
     2101 gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg
     2161 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
     2221 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
     2281 tgctgtctca tcattttggc aaagaattcc tcgagaattc tcacgcgtcc accatgtacc
     2341 catatgatgt tcctgattat gctgggggag caggaggccc taaagatcca gccaaacctc
     2401 cggccaaggc acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg
     2461 tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gggggagcag
     2521 gaggcgaagg tgatgcagtc gaagccattg tggaggagtc cgaaactttt attaaaggaa
     2581 aggagagaaa gacttaccag agacgccggg aagggggcca ggaagaagat gcctgccact
     2641 taccccagaa ccagacggat gggggtgagg tggtccagga tgtcaacagc agtgtacaga
     2701 tggtgatgat ggaacagctg gaccccaccc ttcttcagat gaagactgaa gtaatggagg
     2761 gcacagtggc tccagaagca gaggctgctg tggacgatac ccagattata actttacagg
     2821 ttgtaaatat ggaggaacag cccataaaca taggagaact tcagcttgtt caagtacctg
     2881 ttcctgtgac tgtacctgtt gctaccactt cagtagaaga acttcagggg gcttatgaaa
     2941 atgaagtgtc taaagagggc cttgcggaaa gtgaacccat gatatgccac accctacctt
     3001 tgcctgaagg gtttcaggtg gttaaagtgg gggccaatgg agaggtggag acactagaac
     3061 aaggggaact tccaccccag gaagatccta gttggcaaaa agacccagac tatcagccac
     3121 cagccaaaaa aacaaagaaa accaaaaaga gcaaactgcg ttatacagag gagggcaaag
     3181 atgtagatgt gtctgtctac gattttgagg aagaacagca ggagggtctg ctatcagagg
     3241 ttaatgcaga gaaagtggtt ggtaatatga agcctccaaa gccaacaaaa attaaaaaga
     3301 aaggtgtaaa gaagacattc cagtgtgagc tttgcagtta cacgtgtcca cggcgttcaa
     3361 atttggatcg tcacatgaaa agccacactg atgagagacc acacaagtgc catctctgtg
     3421 gcagggcatt cagaacagtc accctcctga ggaatcacct taacacacac acaggtactc
     3481 gtcctcacaa gtgcccagac tgcgacatgg cctttgtgac cagtggagaa ttggttcggc
     3541 atcgtcgtta caaacacacc cacgagaagc cattcaagtg ttccatgtgc gattacgcca
     3601 gtgtagaagt cagcaaatta aaacgtcaca ttcgctctca tactggagag cgtccgtttc
     3661 agtgcagttt gtgcagttat gccagcaggg acacatacaa gctgaaaagg cacatgagaa
     3721 cccattcagg ggaaaagcct tatgaatgtt atatttgtca tgctcggttt acccaaagtg
     3781 gtaccatgaa gatgcacatt ttacagaagc acacagaaaa tgtggccaaa tttcactgtc
     3841 cccactgtga cacagtcata gcccgaaaaa gtgatttggg tgtccacttg cgaaagcagc
     3901 attcctatat tgagcaaggc aagaaatgcc gttactgtga tgctgtgttt catgagcgct
     3961 atgccctcat ccagcatcag aagtcacaca agaatgagaa gcgctttaag tgtgaccagt
     4021 gtgattacgc ttgtagacag gagaggcaca tgatcatgca caagcgcacc cacaccgggg
     4081 agaagcctta cgcctgcagc cactgcgata agaccttccg ccagaagcag cttctcgaca
     4141 tgcacttcaa gcgctatcac gaccccaact tcgtccctgc ggcttttgtc tgttctaagt
     4201 gtgggaaaac atttacacgt cggaatacca tggcaagaca tgctgataat tgtgctggcc
     4261 cagatggcgt agagggggaa aatggaggag aaacgaagaa gagtaaacgt ggaagaaaaa
     4321 gaaagatgcg ctctaagaaa gaagattcct ctgacagtga aaatgctgaa ccagatctgg
     4381 acgacaatga ggatgaggag gagcctgccg tagaaattga acctgagcca gagcctcagc
     4441 ctgtgacccc agccccacca cccgccaaga agcggagagg acgaccccct ggcagaacca
     4501 accagcccaa acagaaccag ccaacagcta tcattcaggt tgaagaccag aatacaggtg
     4561 caattgagaa cattatagtt gaagtaaaaa aagagccaga tgctgagccc gcagagggag
     4621 aggaagagga ggcccagcca gctgccacag atgcccccaa cggagacctc acgcccgaga
     4681 tgatcctcag catgatggac cgggggggag caggaggcgt cgacggtatc gatcctaaag
     4741 atccagccaa acctccggcc aaggcacaag ttgtgggatg gccaccggtg agatcatacc
     4801 ggaagaacgt gatggtttcc tgccaaaaat caagcggtgg cccggaggcg gcggcgttcg
     4861 tgaagaagct tgatgtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     4921 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     4981 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     5041 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     5101 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     5161 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     5221 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     5281 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     5341 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     5401 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     5461 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     5521 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     5581 tgtacaaggg cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga
     5641 atcctggccc aatgaaaacc ttcaacatct ctcagcagga tctggagctg gtggaggtcg
     5701 ccactgagaa gatcaccatg ctctatgagg acaacaagca ccatgtcggg gcggccatca
     5761 ggaccaagac tggggagatc atctctgctg tccacattga agcctacatt ggcagggtca
     5821 ctgtctgtgc tgaagccatt gccattgggt ctgctgtgag caacgggcag aaggactttg
     5881 acaccattgt ggctgtcagg cacccctact ctgatgaggt ggacagatcc atcagggtgg
     5941 tcagcccctg tggcatgtgt agagagctga tctctgacta tgctcctgac tgctttgtgc
     6001 tcattgagat gaatggcaag ctggtcaaaa ccaccattga ggaactcatc cccctcaagt
     6061 acaccaggaa ctaatgacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     6121 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     6181 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     6241 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     6301 tgctggggat cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg
     6361 tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
     6421 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     6481 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     6541 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     6601 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     6661 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     6721 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     6781 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     6841 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     6901 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     6961 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     7021 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     7081 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     7141 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     7201 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     7261 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     7321 aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc
     7381 ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7441 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7501 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7561 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     7621 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7681 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7741 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7801 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7861 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7921 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7981 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     8041 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtt
//
