LOCUS       sequence-217898-        6478 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6478)
  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 6478)
  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..6478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..885
                     /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             919..1050
                     /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(2466..2485)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2467..2485)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2495..2512)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2495..2511)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2504..2526)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2652..3107)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2789..2810)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3001..3020
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3134..3238
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3239..3307,3308..4099)
                     /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(3457..3476)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4270..4858
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4759..4778
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5012..5029
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5146..5167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5182..5187,5188..5205,5206..5212)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5220..5236
                     /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     5225..5247
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5244..5260
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5244..5260
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5279..5299
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5281..5299
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     5328..5344
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5329..5345
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             6443..6463
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
ORIGIN
        1 gaaatcggta ctggctttcc attcgacccc cattatgtgg aagtcctggg cgagcgcatg
       61 cactacgtcg atgttggtcc gcgcgatggc acccctgtgc tgttcctgca cggtaacccg
      121 acctcctcct acgtgtggcg caacatcatc ccgcatgttg caccgaccca tcgctgcatt
      181 gctccagacc tgatcggtat gggcaaatcc gacaaaccag acctgggtta tttcttcgac
      241 gaccacgtcc gcttcatgga tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg
      301 gtcattcacg actggggctc cgctctgggt ttccactggg ccaagcgcaa tccagagcgc
      361 gtcaaaggta ttgcatttat ggagttcatc cgccctatcc cgacctggga cgaatggcca
      421 gaatttgccc gcgagacctt ccaggccttc cgcaccaccg acgtcggccg caagctgatc
      481 atcgatcaga acgtttttat cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact
      541 gaagtcgaga tggaccatta ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg
      601 tggcgcttcc caaacgagct gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc
      661 gaagaataca tggactggct gcaccagtcc cctgtcccga agctgctgtt ctggggcacc
      721 ccaggcgttc tgatcccacc ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc
      781 aaggctgtgg acatcggccc gggtctgaat ctgctgcaag aagacaaccc ggacctgatc
      841 ggcagcgaga tcgcgcgctg gctgtctact ctggagattt ccggtggtag cggcagcggt
      901 agcactaaga gtgcttgtcc taaagatcca gccaaacctc cggccaaggc acaagttgtg
      961 ggatggccac cggtgagatc ataccggaag aacgtgatgg tttcctgcca aaaatcaagc
     1021 ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg acggagcacc gtacttgagg
     1081 aaaatcgatt tgaggatgta taaagctagc actagttgat gctggggcct tgctcggcac
     1141 caggactatt gggctgtgtt taaacggccc aaatcttaat ttttctcttt tttttctttg
     1201 gctttgggaa cggcataatt ttacaccatt ttaccaaaca tactgagaac gaaaacttca
     1261 aggatgatgt tagaaaaatg tgatttaact agaacttgtt tgatgttagc aaatcatgga
     1321 atgttctaag tctctgaggg tttactgtga agtgttgagg acagtgttga tgcctaacta
     1381 gttttcttag atggaaacag agacattgag ccctctctct tgatcgtaaa ccactccaga
     1441 acggccacgg gtttcccaga gttctatggt cttcccaaga gaatttttaa ttgtaaatgc
     1501 agacttggga aggacttgga cttttaacct ggttttgctt ttgcttttcc tgactccctt
     1561 tgctcagagt cagttgcaca ccagtaggat ggcatgctac gatcagtttc tgtcctgaaa
     1621 gctttgcctc tttcttggca aagtttctgg tatggtcaag cttgtaaata acctttttta
     1681 cattttaatc ttttccatta attaagaggt tgaaaagaag tacagtgtaa gaaaacccag
     1741 cattttaatt atttgcaaat taaggtgcca cagacacaga cttagtggta tgtaaatgtt
     1801 gactcggatc acaatcatgt agcagagggc actgggcctg ttgcctgcca gtcatggaca
     1861 cccatgtgaa aaatcctgat ttccagctca tggagccagc gtttgaacct tgaacaaata
     1921 aatttcattt atgattttct catcaacatt ttctttgctc tgtttgtagc tgaatgttac
     1981 atttttttaa aaatctctgt tcaagccaga agagttgatt atgagtggcc acagcaaccc
     2041 tggcaagctg ggccaaaaaa tttcagtagg tcagtaattt aaacttcaca tctggaaaaa
     2101 aaattattaa taatgtgaaa caaaccaact taaaaagtga ttcttgcaca tgaattgtca
     2161 catattatat gtgtttttta aagagcctca gtctgactga tttcaaacac tttttttctc
     2221 ctgtgtattg cttttaagag agccatcagt tagctatcag aatctaggtt gatgcatttt
     2281 tgtacttagc tgtactgtgt gatatttttc attattttag gatgccaaca tgagacctgt
     2341 aataaaatat gtaatggggt tgaaggctgg ggaggaggat ctactgctgt acagctaata
     2401 aatcataacg gattaacagg caactagttc tagagcggcc gccaccgcgg tggagctcca
     2461 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     2521 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     2581 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2641 atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2701 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2761 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2821 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2881 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2941 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3001 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3061 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     3121 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3181 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3241 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3301 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3361 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3421 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3481 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3541 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3601 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3661 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3721 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3781 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3841 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3901 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3961 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4021 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4081 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4141 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4201 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4261 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4321 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4381 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4441 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4501 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4561 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4621 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4681 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4741 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4801 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4861 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4921 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4981 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5041 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5101 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     5161 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     5221 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
     5281 aattaaccct cactaaaggg aacaaaagct gggtaccggg ccccccctcg aggtcgacgg
     5341 tatcgataag cttgatatcg aattcgggta ctaatgtcct gcaagatggg tgtgctgtta
     5401 ttttgggagc gattataaaa caaccaaggt cacagtcaca tggcagagct gacatggtta
     5461 taagtagaaa agagccaggc tttcagggaa tgaagggctg aagcaacgaa tagactgaga
     5521 cagttctgct ggaggaactc aggcccctaa agccgggagt gctattacca gccctaatcc
     5581 caacacttgg gaggctgagg ctggcctggg ctacatggaa agaagaaggg aaggggaaac
     5641 cattcaggtg aaaggattgt cccaggagtg ttccctttct agcacagtgc ctggatgtag
     5701 tgggtcttag atcgcctcaa agcagccttt tcttctcttt aaattcaggg cttaatctat
     5761 aattatgctc cctgagcgcc atgtccaaat gtccatcaca gaacaggggg ataactctgc
     5821 atctagaacc gcccagtcat ttcacctaca cacaactatt tcagagcagt acagggtcag
     5881 aaatgtcaac tgcatcatat actctgtgaa atactccttg tcatgtgcag tctagggctg
     5941 gatttttttt tccctgccct gcattactat acatcctaaa tacagaacat aaaggaaaac
     6001 taaaaatgtt aaagtggagg cctgtgaggt aaatgtcagt atttggtatc tgacaatttc
     6061 tagccttttg ggattttatg tgtggccact taacgttcgc agggctgttt tgtttctgct
     6121 gacttgggca tcactgctga ggctttcttg ttgctgcatc ccattcattg tcagcatcgg
     6181 gaacaatgcc tgtgctcgct gggggcttta atgtacgtac cctttgtttt gttcctgccc
     6241 ttctttgcca gcaacagcca tcattcaggt cgaagatcag aatacaggtg caattgagaa
     6301 cattatagtt gaagtcaaaa aggagccaga tgccgagcct gcggaggggg aagaagagga
     6361 ggctcaggca gccaccacag acgcccccaa cggagacctc acgcctgaga tgatcctcag
     6421 catgatggac cgggaattca tggaggatct gtactttcag agcgataacg atggatcc
//
