LOCUS       sequence-292662-        6994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CTCF tagging with mAID-Clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6994)
  AUTHORS   Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano 
            R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, 
            Hayashi KI, Kanemaki MT
  TITLE     The auxin-inducible degron 2 technology provides sharp degradation 
            control in yeast, mammalian cells, and mice.
  JOURNAL   Nat Commun. 2020 Nov 11;11(1):5701. doi: 10.1038/s41467-020-19532-z.
  PUBMED    33177522
REFERENCE   2  (bases 1 to 6994)
  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 
            Commun."; date: "2020-11-11"; volume: "11(1):5701. doi"; pages: " 
            10.1038/s41467-020-19532-z"
COMMENT     Sequence Label: CTCF-mAC donor (Neo)
FEATURES             Location/Qualifiers
     source          1..6994
                     /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     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     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1115..1318
                     /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(1325..1327,1328..1330,1331..2044)
                     /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(1370..1391)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1978..1999
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2162..2283
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2199..2218)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2253..2272
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2290..2745)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2772..2876
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2878..3235
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2878..3069
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2901..2921)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3086..3221
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3270..4064
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3270..4064
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3324..3343)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3934..3953
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4252..4271)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(4806..4826)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4806..4824)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4845..4861)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4845..4861)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4858..4880)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4869..4885
                     /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(4893..4899,4900..4917,4918..4923))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4938..4959
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5076..5093)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5247..5835)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5327..5346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6006..6797,6798..6866))
                     /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     6629..6648
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6867..6971)
                     /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 gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctctg
      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 gaccggggat ccggtgcagg cgccaaggag aagagtgctt gtcctaaaga
     1141 tccagccaaa cctccggcca aggcacaagt tgtgggatgg ccaccggtga gatcataccg
     1201 gaagaacgtg atggtttcct gccaaaaatc aagcggtggc ccggaggcgg cggcgttcgt
     1261 gaaggtatca atggacggag caccgtactt gaggaaaatc gatttgagga tgtataaagc
     1321 tagcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1381 ggacggcgac gtaaacggcc acaagttcag cgtccgcggc gagggcgagg gcgatgccac
     1441 caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1501 caccctcgtg accaccttcg gctacggcgt ggcctgcttc agccgctacc ccgaccacat
     1561 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1621 ctctttcaag gacgacggta cctacaagac ccgcgccgag gtgaagttcg agggcgacac
     1681 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1741 gcacaagctg gagtacaact tcaacagcca caacgtctat atcacggccg acaagcagaa
     1801 gaacggcatc aaggctaact tcaagatccg ccacaacgtt gaggacggca gcgtgcagct
     1861 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1921 ccactacctg agccatcagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     1981 ggtcctgctg gagttcgtga ccgccgccgg gattacacat ggcatggacg agctgtacaa
     2041 gtaatctaga taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt
     2101 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2161 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2221 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2281 ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2341 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2401 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     2461 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     2521 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2581 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2641 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2701 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     2761 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2821 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     2881 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2941 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3001 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3061 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3121 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3181 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     3241 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3301 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3361 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3421 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     3481 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3541 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3601 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3661 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3721 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3781 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3841 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3901 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3961 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4021 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4081 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4141 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4201 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     4261 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     4321 gcacggtgtt gggtcgttgg atccgcgtcg ccaggacttc tctgggctgt gtttaaacgg
     4381 cccgcatctt aatttttctc ccttctttct ttttttggct ttgggaaaag catcatttta
     4441 ccaaacatac cgagaacgaa aacttcaagg atgatgttag aaaaaaatgt gatttaacta
     4501 gaacttgctg tctgatgtta gcaaatcatg gaatgttctg agtccctgag ggtttactgt
     4561 gaagtgctga ggacagtgtt gacaactaac tcgttttcct agatggaaac ggagacattg
     4621 acccctccct ccatgtggta aaccactcca gaatggccac caggcttccc agagttctat
     4681 ggtcttcttc ccaagagagt ttttaattgt aaatgcatac ttgggaagga cttagagttt
     4741 taaactgttt tttgcttttg cttttccctg actccctttg cttggagggt acccagcttt
     4801 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     4861 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccgggag cataaagtgt
     4921 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4981 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5041 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5101 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5161 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5221 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5281 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5341 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5401 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5461 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5521 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5581 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5641 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5701 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5761 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5821 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5881 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5941 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6001 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6061 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6121 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6181 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6241 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6301 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6361 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6421 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6481 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6541 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6601 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6661 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6721 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6781 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6841 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6901 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6961 ggggttccgc gcacatttcc ccgaaaagtg ccac
//
