LOCUS       sequence-213345-        6479 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for knock-in mAID-Halo fusion to the c-terminal of 
            mouse Ctcf coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6479)
  AUTHORS   Gu B, Posfai E, Rossant J
  TITLE     Efficient generation of targeted large insertions by microinjection 
            into two-cell-stage mouse embryos.
  JOURNAL   Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166.
  PUBMED    29889212
REFERENCE   2  (bases 1 to 6479)
  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 
            Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166."
COMMENT     Sequence Label: Ctcf-Halo-mAID donor
FEATURES             Location/Qualifiers
     source          1..6479
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(16..35)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     226..247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        410..514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(515..583,584..1375)
                     /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(733..752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1546..2134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2035..2054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2288..2305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2422..2443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2458..2463,2464..2481,2482..2488)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2496..2512
                     /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     2501..2523
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2520..2536
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2520..2536
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2555..2575
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2557..2575
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2604..2620
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2605..2621
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3719..3739
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             3755..4639
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             4673..4804
                     /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"
     primer_bind     complement(6220..6239)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6221..6239)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6249..6266)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6249..6265)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6258..6280)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
       61 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      121 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      181 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
      241 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
      301 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
      361 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      481 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
      541 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
      601 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
      661 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
      721 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
      781 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
      841 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
      901 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
      961 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1021 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     1081 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1141 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1201 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     1261 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     1321 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     1381 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     1441 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     1501 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1561 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     1621 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     1681 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     1741 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     1801 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     1861 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     1921 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     1981 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2041 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2101 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2161 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2221 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2281 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     2341 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2401 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     2461 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     2521 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     2581 aaagctgggt accgggcccc ccctcgaggt cgacggtatc gataagcttg atatcgaatt
     2641 cgggtactaa tgtcctgcaa gatgggtgtg ctgttatttt gggagcgatt ataaaacaac
     2701 caaggtcaca gtcacatggc agagctgaca tggttataag tagaaaagag ccaggctttc
     2761 agggaatgaa gggctgaagc aacgaataga ctgagacagt tctgctggag gaactcaggc
     2821 ccctaaagcc gggagtgcta ttaccagccc taatcccaac acttgggagg ctgaggctgg
     2881 cctgggctac atggaaagaa gaagggaagg ggaaaccatt caggtgaaag gattgtccca
     2941 ggagtgttcc ctttctagca cagtgcctgg atgtagtggg tcttagatcg cctcaaagca
     3001 gccttttctt ctctttaaat tcagggctta atctataatt atgctccctg agcgccatgt
     3061 ccaaatgtcc atcacagaac agggggataa ctctgcatct agaaccgccc agtcatttca
     3121 cctacacaca actatttcag agcagtacag ggtcagaaat gtcaactgca tcatatactc
     3181 tgtgaaatac tccttgtcat gtgcagtcta gggctggatt tttttttccc tgccctgcat
     3241 tactatacat cctaaataca gaacataaag gaaaactaaa aatgttaaag tggaggcctg
     3301 tgaggtaaat gtcagtattt ggtatctgac aatttctagc cttttgggat tttatgtgtg
     3361 gccacttaac gttcgcaggg ctgttttgtt tctgctgact tgggcatcac tgctgaggct
     3421 ttcttgttgc tgcatcccat tcattgtcag catcgggaac aatgcctgtg ctcgctgggg
     3481 gctttaatgt acgtaccctt tgttttgttc ctgcccttct ttgccagcaa cagccatcat
     3541 tcaggtcgaa gatcagaata caggtgcaat tgagaacatt atagttgaag tcaaaaagga
     3601 gccagatgcc gagcctgcgg agggggaaga agaggaggct caggcagcca ccacagacgc
     3661 ccccaacgga gacctcacgc ctgagatgat cctcagcatg atggaccggg aattcatgga
     3721 ggatctgtac tttcagagcg ataacgatgg atccgaaatc ggtactggct ttccattcga
     3781 cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga
     3841 tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat
     3901 catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa
     3961 atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt
     4021 catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct
     4081 gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt
     4141 catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga ccttccaggc
     4201 cttccgcacc accgacgtcg gccgcaagct gatcatcgat cagaacgttt ttatcgaggg
     4261 tacgctgccg atgggtgtcg tccgcccgct gactgaagtc gagatggacc attaccgcga
     4321 gccgttcctg aatcctgttg accgcgagcc actgtggcgc ttcccaaacg agctgccaat
     4381 cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca
     4441 gtcccctgtc ccgaagctgc tgttctgggg caccccaggc gttctgatcc caccggccga
     4501 agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct
     4561 gaatctgctg caagaagaca acccggacct gatcggcagc gagatcgcgc gctggctgtc
     4621 tactctggag atttccggtg gtagcggcag cggtagcact aagagtgctt gtcctaaaga
     4681 tccagccaaa cctccggcca aggcacaagt tgtgggatgg ccaccggtga gatcataccg
     4741 gaagaacgtg atggtttcct gccaaaaatc aagcggtggc ccggaggcgg cggcgttcgt
     4801 gaaggtatca atggacggag caccgtactt gaggaaaatc gatttgagga tgtataaagc
     4861 tagcactagt tgatgctggg gccttgctcg gcaccaggac tattgggctg tgtttaaacg
     4921 gcccaaatct taatttttct cttttttttc tttggctttg ggaacggcat aattttacac
     4981 cattttacca aacatactga gaacgaaaac ttcaaggatg atgttagaaa aatgtgattt
     5041 aactagaact tgtttgatgt tagcaaatca tggaatgttc taagtctctg agggtttact
     5101 gtgaagtgtt gaggacagtg ttgatgccta actagttttc ttagatggaa acagagacat
     5161 tgagccctct ctcttgatcg taaaccactc cagaacggcc acgggtttcc cagagttcta
     5221 tggtcttccc aagagaattt ttaattgtaa atgcagactt gggaaggact tggactttta
     5281 acctggtttt gcttttgctt ttcctgactc cctttgctca gagtcagttg cacaccagta
     5341 ggatggcatg ctacgatcag tttctgtcct gaaagctttg cctctttctt ggcaaagttt
     5401 ctggtatggt caagcttgta aataaccttt tttacatttt aatcttttcc attaattaag
     5461 aggttgaaaa gaagtacagt gtaagaaaac ccagcatttt aattatttgc aaattaaggt
     5521 gccacagaca cagacttagt ggtatgtaaa tgttgactcg gatcacaatc atgtagcaga
     5581 gggcactggg cctgttgcct gccagtcatg gacacccatg tgaaaaatcc tgatttccag
     5641 ctcatggagc cagcgtttga accttgaaca aataaatttc atttatgatt ttctcatcaa
     5701 cattttcttt gctctgtttg tagctgaatg ttacattttt ttaaaaatct ctgttcaagc
     5761 cagaagagtt gattatgagt ggccacagca accctggcaa gctgggccaa aaaatttcag
     5821 taggtcagta atttaaactt cacatctgga aaaaaaatta ttaataatgt gaaacaaacc
     5881 aacttaaaaa gtgattcttg cacatgaatt gtcacatatt atatgtgttt tttaaagagc
     5941 ctcagtctga ctgatttcaa acactttttt tctcctgtgt attgctttta agagagccat
     6001 cagttagcta tcagaatcta ggttgatgca tttttgtact tagctgtact gtgtgatatt
     6061 tttcattatt ttaggatgcc aacatgagac ctgtaataaa atatgtaatg gggttgaagg
     6121 ctggggagga ggatctactg ctgtacagct aataaatcat aacggattaa caggcaacta
     6181 gttctagagc ggccgccacc gcggtggagc tccaattcgc cctatagtga gtcgtattac
     6241 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     6301 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     6361 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     6421 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccag
//
