LOCUS       sequence-217585-        5902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for knock-in HaloTag fusing to the c-terminal of mouse
            Id2 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5902)
  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 5902)
  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: Id2-Halo donor
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1030..1914)
                     /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             complement(1930..1950)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(2969..2985)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2970..2986)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3015..3035)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3015..3033)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3054..3070)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3054..3070)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3067..3089)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3078..3094
                     /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(3102..3108,3109..3126,3127..3132))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3147..3168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3285..3302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3456..4044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3536..3555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4215..5006,5007..5075))
                     /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     4838..4857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5076..5180)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5207..5662
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5294..5313)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5504..5525
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5788..5810
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5802..5819
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5803..5819
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5829..5848
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5829..5847
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agtatataaa gaaaaccttc caactgtaga aagggcactg aaagtctctc ataaataacg
       61 gtatcacagt ccaggccgga gaacgacacc tgggcaagac gatcatcctt agttttcctt
      121 ccgctttctt tttaaaaagg tccattcaac gtgttctcct ggtgaaatgg ctgataacaa
      181 aagtcttaaa agatcttgta gacttcttct tgtgcaaaag aaaaagagaa agtaaaaaga
      241 aaaaaaaaag tccccaaatg cctgcaaaag attgggagag aaaggcagtt taggagaatg
      301 taacctaagg gcagaggcgg gcgagcctgg cgccgaggaa gccacagttt gagcatttaa
      361 ccaaaccctg gcgtccagat gcggcacaca tacccggccc cagcagcaca aattaaacat
      421 gaaaatagcg gagactccag gggaaaactt cccaaaccca gtctcctatt tggatctcta
      481 acagcccgct cacaggcgcc agtctgcttc ttgtaacaat tatttgattg aaaacagccc
      541 tttagttaac agatatggaa atctggatcc cagagcacag gggtaaggct cggggtagcc
      601 gtcttcagct agccagagaa atctcagagg actacagttt tgggaaaaga gaatcactat
      661 ggaatttgtg gcgataagat taattacagc tgaggttacc acctcagtgt accgccactg
      721 ggctcataaa agtcagccac acttggtcct gacaccttca tagtaagtta ctggcatttc
      781 ttttctttct tttttttctt tcttttctgt ttttgtaaag gcaactgttc tgtagtcctg
      841 agcaaccaca gctacaagcc ccttcatgcc tatgtggtaa gtcaagcggc acccgtaaca
      901 cagtgaagta agcatgcatt taccaaacca aacatttaac agacacacaa gcacattacc
      961 gaaacgcgca cacaggaggc acctgcaaca ctcaccattt atttaactag tgctaccgct
     1021 gccgctacca ccggaaatct ccagagtaga cagccagcgc gcgatctcgc tgccgatcag
     1081 gtccgggttg tcttcttgca gcagattcag acccgggccg atgtccacag ccttgcagtt
     1141 aggcaggctt ttggccaggc gagcggcttc ggccggtggg atcagaacgc ctggggtgcc
     1201 ccagaacagc agcttcggga caggggactg gtgcagccag tccatgtatt cttcgaccag
     1261 cgcgacgatg ttcgctggct caccggcgat tggcagctcg tttgggaagc gccacagtgg
     1321 ctcgcggtca acaggattca ggaacggctc gcggtaatgg tccatctcga cttcagtcag
     1381 cgggcggacg acacccatcg gcagcgtacc ctcgataaaa acgttctgat cgatgatcag
     1441 cttgcggccg acgtcggtgg tgcggaaggc ctggaaggtc tcgcgggcaa attctggcca
     1501 ttcgtcccag gtcgggatag ggcggatgaa ctccataaat gcaatacctt tgacgcgctc
     1561 tggattgcgc ttggcccagt ggaaacccag agcggagccc cagtcgtgaa tgaccaggac
     1621 gacctcttcc agacccaggg cttcgatgaa ggcatccatg aagcggacgt ggtcgtcgaa
     1681 gaaataaccc aggtctggtt tgtcggattt gcccataccg atcaggtctg gagcaatgca
     1741 gcgatgggtc ggtgcaacat gcgggatgat gttgcgccac acgtaggagg aggtcgggtt
     1801 accgtgcagg aacagcacag gggtgccatc gcgcggacca acatcgacgt agtgcatgcg
     1861 ctcgcccagg acttccacat aatgggggtc gaatggaaag ccagtaccga tttcggatcc
     1921 atcgttatcg ctctgaaagt acagatcctc catgaattcg atatcgccac agagtacttt
     1981 gctatcattc gacataagct cagaagggaa ttcagatgcc tggggagaaa gaggataaaa
     2041 ggcaggatat taggtaatct taaaggaagg tagcccccct cctcctttca caaaagtgcc
     2101 aacaagtccc atgaaacagg atcatgctac agcgcagatc cagatgcaac ccacacagaa
     2161 cttataagta gcgatttcac atgacgctca atatacagtt tcctcaaaaa taaagttaat
     2221 gaacctgtct ctacgatggg ctttaagagt gacagctcgg gagacccgga gcgtctgggc
     2281 agtggcgtac ttgcaagcta gtctcacctg caaggacagg atgctgatgt ccgtgttcag
     2341 ggtggtcagc ggcgtcctgg acgcctggtt ctgtccaggt ctctggtgat gcaggctgac
     2401 gatagtggga tgcgagtcca gggcgatctg caggtccaag atgtaatcga tgacgtgctg
     2461 caggatttcc atcttggtca ccttcttgtt ctgggggatg ctgggcacca gttccttgag
     2521 cttggagtag cagtcgttca tgttgtagag cagactcatc gggtcgtcca ccggggtttt
     2581 gctccgggag atgcccaagc tgtggtccga caggctgttt ttcctaacgg acctcaccgg
     2641 actgaaggct ttcatgctgc tcgtaggagg aagaccggga gggagccgcc acagggggct
     2701 gagctgactg cgcggcacca ggctcggttc agaatgaagc gcgagccggg cggggcgtct
     2761 tttatgtgca ctcgccctgg ccgctcaatc gggcatccct gaattgccat tggtggaagg
     2821 gctcccgtcc atcataccag gcgaattctc ccattggtga gtatgagctc tcggttcttc
     2881 cgcgttcgca gccaatgcct gtagggtggg aggagcgggc gagagcccag ctggggtggc
     2941 aaattgagta cagtgtgatc aagcttatcg ataccgtcga cctcgagggg gggcccggta
     3001 cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     3061 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3121 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3181 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3241 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3301 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3361 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3421 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3481 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3541 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3601 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3661 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3721 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3781 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3841 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3901 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3961 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4021 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4081 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4141 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4201 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4261 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4321 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4381 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4441 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4501 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4561 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4621 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4681 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4741 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4801 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4861 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4921 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4981 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5041 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5101 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5161 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc
     5221 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5281 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5341 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5401 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5461 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5521 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5581 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5641 aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc
     5701 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     5761 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     5821 agcgcgcgta atacgactca ctatagggcg aattggagct ccaccgcggt ggcggccgct
     5881 ctagaactag catggtggga at
//
