LOCUS       sequence-310780-        5441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knockin donor vector of miniAID-mClover3 to C-terminus of endogenous
            human CTCF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5441)
  AUTHORS   Hyle J, Zhang Y, Wright S, Xu B, Shao Y, Easton J, Tian L, Feng R, 
            Xu P, Li C
  TITLE     Acute depletion of CTCF directly affects MYC regulation through loss
            of enhancer-promoter looping.
  JOURNAL   Nucleic Acids Res. 2019 Jul 26;47(13):6699-6713. doi: 
            10.1093/nar/gkz462.
  PUBMED    31127282
REFERENCE   2  (bases 1 to 5441)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2019-07-26- 26"; volume: "47"; issue: 
            "13"; pages: "6699-6713"
COMMENT     Sequence Label: pKnockinDonor-humanCTCF-miniAID-mClover3
FEATURES             Location/Qualifiers
     source          1..5441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     240..260
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             1097..1300
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(1307..1309,1310..1312,1313..2026)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1352..1373)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1960..1981
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2868..2887)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2869..2887)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2897..2914)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2897..2913)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2906..2928)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3055..3510
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3142..3161)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3352..3373
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3536..3640
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3641..3709,3710..4501)
                     /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     complement(3859..3878)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4672..5260
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5161..5180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5414..5431
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
      241 caattaaccc tcactaaagg gaacaaaagc tggccggtga tggcggagcc ttgtgcagta
      301 gaagggtact tgtgttctgc aagatgggta ctgttaatgt gggcgggttt tgaaaaacaa
      361 ccaaagtcac agtcagctgg cagaatggat gcggtaatga atataaaaga gccaaatcgt
      421 aaaggttgga gagggaaagc agtttctgca ctgagattgc cccttctaag aaactcaggg
      481 tcaggcggtg gctcatgcct gtgatcccag cactttgggg ggccaaggcg ggaggatcac
      541 ctgaggtcag tagttccata ccagcctggt caacatggtg aaaccccatc tctgctaaaa
      601 atacaaaaat tagccaggcg taggggtggg cacctgtagt cccatctact caggaggctg
      661 aggcaggaga attgctttaa cccggggggc agaggttgca gtgagctgag atcatgcccg
      721 tgcactccag cctggtcata gagcaagact ccgtctcaaa aaataaaata aaaaataaat
      781 tgactgtctc tggaccgcta tctaataagg ctgtcctgca ttgctgacat cccgttcgct
      841 gtcagtctaa aagacccttg tgattcttgg ggctttaatg gaccatttgt tctgtctgtg
      901 ctcttctttg ccagcaacag ctatcattca ggttgaagac cagaatacag gtgcaattga
      961 gaacattata gttgaagtaa aaaaagagcc agatgctgag cccgcagagg gagaggaaga
     1021 ggaggcccag ccagctgcca cagatgcccc caacggagac ctcacgcccg agatgatcct
     1081 cagcatgatg gaccggaagg agaagagtgc ttgtcctaaa gatccagcca aacctccggc
     1141 caaggcacaa gttgtgggat ggccaccggt gagatcatac cggaagaacg tgatggtttc
     1201 ctgccaaaaa tcaagcggtg gcccggaggc ggcggcgttc gtgaaggtat caatggacgg
     1261 agcaccgtac ttgaggaaaa tcgatttgag gatgtataaa gctagcatgg tgagcaaggg
     1321 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1381 ccacaagttc agcgtccgcg gcgagggcga gggcgatgcc accaacggca agctgaccct
     1441 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt
     1501 cggctacggc gtggcctgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1561 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atctctttca aggacgacgg
     1621 tacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1681 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1741 cttcaacagc cacaacgtct atatcacggc cgacaagcag aagaacggca tcaaggctaa
     1801 cttcaagatc cgccacaacg ttgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1861 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagccatca
     1921 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1981 gaccgccgcc gggattacac atggcatgga cgagctgtac aagtaagtac aagtaatgat
     2041 ggcggagcct tgtgcgtcgc caggactgct ctgggctgtg tttaaacggc ccgcatctta
     2101 atttttctcc cttctttctt tttttggctt tgggaaaagc atcattttac caaacatacc
     2161 gagaacgaaa acttcaagga tgatgttaga aaaaaatgtg atttaactag aacttgctgt
     2221 ctgatgttag caaatcatgg aatgttctga gtccctgagg gtttactgtg aagtgctgag
     2281 gacagtgttg acaactaact cgttttccta gatggaaacg gagacattga cccctccctc
     2341 catgtggtaa accactccag aatggccacc aggcttccca gagttctatg gtcttcttcc
     2401 caagagagtt tttaattgta aatgcatact tgggaaggac ttagagtttt aaactgtttt
     2461 ttgcttttgc ttttccctga ctccctttgc ttggagtcag ctgcacacca gtagtatggc
     2521 atgctacgat caggttctgt cctgaaagct ttgcctcttt cttggcaaag tttctggtat
     2581 ggtcaagctt gtaaataact ttttttacat tttaatcttt tccattaatt aagaggttga
     2641 aaagaagtgc agtgtaagaa aacccagcat tttaattact tgcaaattaa gttaccacag
     2701 actctgtagt gtgtaaatgt tgacaaggaa ttggatcaca atcatgtagc agaatggcac
     2761 ccagaccact gcccaccagt gacggacatg cacgtggcag atcatgattt ccagcccacg
     2821 gagccagcat ttgaacgcac aaggctccgc catcaccggc caattcgccc tatagtgagt
     2881 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     2941 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     3001 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
     3061 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3121 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3181 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3241 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3301 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3361 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3421 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3481 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     3541 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3601 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3661 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3721 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3781 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3841 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3901 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3961 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4021 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4081 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4141 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     4201 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4261 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4321 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4381 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4441 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4501 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4561 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4621 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4681 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4741 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4801 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4861 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4921 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4981 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5041 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5101 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5161 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5221 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5281 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5341 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5401 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
