LOCUS       sequence-292657-        7263 bp    DNA     circular SYN 14-APR-2025
DEFINITION  CTCF tagging with mAID-clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7263)
  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 7263)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 (Hygro)
FEATURES             Location/Qualifiers
     source          1..7263
                     /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     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     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"
     protein_bind    2314..2347
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2408..2907
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2443..2466)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2840..2859
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2968..3987
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2968..3987
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     protein_bind    4550..4583
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5075..5095)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5075..5093)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5114..5130)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5114..5130)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5127..5149)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5138..5154
                     /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(5162..5168,5169..5186,5187..5192))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5207..5228
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5345..5362)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5516..6104)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5596..5615)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6275..7066,7067..7135))
                     /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     6898..6917
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7136..7240)
                     /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 ttaacgcgtc gatcatattc aataaccctt aatataactt cgtataatgt atgctatacg
     2341 aagttattag gtctgaagag gagtttacgt ccagccaagc ttaggatctc gacctcgaaa
     2401 ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc
     2461 ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc
     2521 ggtaggcgcc aaccgactcc gttctttggt ggccccttcg cgccaccttc tactcctccc
     2581 ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa
     2641 gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta
     2701 ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg
     2761 ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga
     2821 aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc
     2881 tcctcttcct catctccggg cctttcgacc tgcatccatc tagatctcga gcagctgaag
     2941 cttaccgcta gcatggatag atccggaaag cctgaactca ccgcgacgtc tgtcgagaag
     3001 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
     3061 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
     3121 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
     3181 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
     3241 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
     3301 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     3361 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     3421 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     3481 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     3541 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     3601 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     3661 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     3721 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     3781 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     3841 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     3901 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     3961 agcactcgtc cgagggcaaa ggaataggct agccgcccgc cccacgaccc gcagcgcccg
     4021 accgaaagga gcgcacgacc ccatgcatcg atgatatcag atccccggga tgcagaaatt
     4081 gatgatctat taaacaataa agatgtccac taaaatggaa gtttttcctg tcatactttg
     4141 ttaagaaggg tgagaacaga gtacctacat tttgaatgga aggattggag ctacgggggt
     4201 gggggtgggg tgggattaga taaatgcctg ctctttactg aaggctcttt actattgctt
     4261 tatgataatg tttcatagtt ggatatcata atttaaacaa gcaaaaccaa attaagggcc
     4321 agctcattcc tcccactcat gatctataga tctatagatc tctcgtggga tcattgtttt
     4381 tctcttgatt cccactttgt ggttctaagt actgtggttt ccaaatgtgt cagtttcata
     4441 gcctgaagaa cgagatcagc agcctctgtt ccacatacac ttcattctca gtattgtttt
     4501 gccaagttct aattccatca gaagctggtc gagatccgga acccttaata taacttcgta
     4561 taatgtatgc tatacgaagt tattaggtcc ctcgaagagg ttcactagga tccgcgtcgc
     4621 caggacttct ctgggctgtg tttaaacggc ccgcatctta atttttctcc cttctttctt
     4681 tttttggctt tgggaaaagc atcattttac caaacatacc gagaacgaaa acttcaagga
     4741 tgatgttaga aaaaaatgtg atttaactag aacttgctgt ctgatgttag caaatcatgg
     4801 aatgttctga gtccctgagg gtttactgtg aagtgctgag gacagtgttg acaactaact
     4861 cgttttccta gatggaaacg gagacattga cccctccctc catgtggtaa accactccag
     4921 aatggccacc aggcttccca gagttctatg gtcttcttcc caagagagtt tttaattgta
     4981 aatgcatact tgggaaggac ttagagtttt aaactgtttt ttgcttttgc ttttccctga
     5041 ctccctttgc ttggagggta cccagctttt gttcccttta gtgagggtta attgcgcgct
     5101 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5161 acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5221 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5281 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5341 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5401 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5461 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5521 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5581 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5641 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5701 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5761 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5821 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5881 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5941 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6001 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6061 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6121 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6181 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6241 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6301 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6361 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6421 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6481 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6541 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6601 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6661 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6721 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6781 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6841 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6901 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6961 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7021 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7081 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7141 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7201 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7261 cac
//
