LOCUS       sequence-213388-        7323 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for knock-in T2A-H2B-Halo to the c-terminal of mouse 
            Fgfr2 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7323)
  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 7323)
  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: Fgfr2-T2A-H2B-Halo donor
FEATURES             Location/Qualifiers
     source          1..7323
                     /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             4620..4673
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4680..5057
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      5070..5079
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5079..5099
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             5115..5999
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(6004..6020)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6004..6020)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(7064..7083)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7065..7083)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7093..7110)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7093..7109)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7102..7124)
                     /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 atatctgctg
     2641 atgttgttgg agccgataca tcccccttct tggatggcga ctctttctct tgccaaccct
     2701 agtgatgaat tatagtgggt ttttgtcttt taaaccctat gatcgccaac attccatggg
     2761 ctctctgctg ataatgctga ggctgtttgt gctgtagtct gcgctttttg ccccctctca
     2821 gaaaaactgt atgtcatagg agttgctggc tattgggtac ataagcaaag ccaccctatt
     2881 gtgccagtgc cttagacagt gagacaagaa aggcccctgg ttagaaatct tatcaggact
     2941 gggaatgtaa ctcagttgat aagagtgctt gcttagcgtg cacacagccc tgggttcaac
     3001 cgcctagtac tacagaaact gagtgtggct tcacacacct gtaatcccag cacttggaga
     3061 gatagatgca ggaggattag aagttcaagg ttatctttag tcacatagta ttggtagcca
     3121 gccagcctgg aatacttgag atacttacag gaaggaagga aggaaggaaa gaaggaggga
     3181 gagaggacag gaggaaggag atagatatac acagaaagag acagagaaac agagattcag
     3241 gagacacaaa gacatacgga gacacagtga gagaaacaga aagacacaga gagagacaga
     3301 gagacataaa gagaaatccc ttctagtttt gtccagggtc agttagtaag tactttctgt
     3361 aaggcccaaa gagtaaacat tttcagcttc aaggccatat ggtctctgtc gaagctgcca
     3421 ttctagcatg aaagcagcca cagctctatg taaataaaga gggtctgtgt tccactaaac
     3481 tttatggact ctgaaactgg gaaatttata taatttcata taaatttata tcatttcatg
     3541 tcattggctg ccaaaagata tgactcaatt tgatttttct actagccatg tacaaatgtt
     3601 gagaagtcgc cttagttcat ggaccaaaca gaaacacaca tgggtttgag ccttgtcctg
     3661 agctggagga agagagtgac ccaaagggac cttggtagca gccagggatg tgttggggag
     3721 cagagaaact tttatgaact tcagtttcag tactgaaact tccctttccc tagacttcct
     3781 ttgacagccc attacgggct taacatccat gagggtagcc acacctttcc ctaacccgtt
     3841 tctgttgctt actctggagc tcctgagggt aatggattat atctgttaaa ctgtgcaaca
     3901 aacggagatg acttctcccc taaatgatcc tagaaatgac aactgtagaa tgtgattccg
     3961 aaaatacata ttcatgtcct ggctttgtaa agggtcctgc tgggtttact tcactgggtc
     4021 ttaaactccg attgtgagcc gtagtgaaga gggctgtata tagtgggtca ccgaggtcct
     4081 gtagcagagt gggcaagctc actgcctgct accagcagtt cactatgttt tatggtctgc
     4141 tgcctgctgg tggtttatag atgctgtgtc gtaagagaaa agttcagggt agcctggagt
     4201 gaatggagtt ggggtatcag ggaggtcttt gtacactggg gtgagctagg cctctggaaa
     4261 gcttcctggg ggttccccgt cccttcccaa atctgataga gatttttcag gagcttctgt
     4321 ttaaaatgag attcttctct tcccttcttg caggaatact tggacctcag tcagcctctc
     4381 gaaccgtatt caccttgtta tcctgaccca aggtgaaata aaacgtctct cttcccttct
     4441 tgcaggaata cttggatctc acccagcctc tcgaacagta ttctcctagt taccccgaca
     4501 caaggagctc ttgttcttca ggggacgatt ctgtgttttc tccagacccc atgccttatg
     4561 aaccctgtct gcctcagtat ccacacataa acggcagtgt taaaacagat atcgaattcg
     4621 agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc cctaccggta
     4681 tgccagagcc agcgaagtct gctcccgccc cgaaaaaggg ctccaagaag gcggtgacta
     4741 aggcgcagaa gaaaggcggc aagaagcgca agcgcagccg caaggagagc tattccatct
     4801 atgtgtacaa ggttctgaag caggtccacc ctgacaccgg catttcgtcc aaggccatgg
     4861 gcatcatgaa ttcgtttgtg aacgacattt tcgagcgcat cgcaggtgag gcttcccgcc
     4921 tggcgcatta caacaagcgc tcgaccatca cctccaggga gatccagacg gccgtgcgcc
     4981 tgctgctgcc tggggagttg gccaagcacg ccgtgtccga gggtactaag gccatcacca
     5041 agtacaccag cgctaaggat ccaccggtcg ccaccatgga ggatctgtac tttcagagcg
     5101 ataacgatgg atccgaaatc ggtactggct ttccattcga cccccattat gtggaagtcc
     5161 tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc
     5221 tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga
     5281 cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg
     5341 gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg
     5401 aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc
     5461 gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct
     5521 gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg
     5581 gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg
     5641 tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg
     5701 accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca
     5761 tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc
     5821 tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa
     5881 gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca
     5941 acccggacct gatcggcagc gagatcgcgc gctggctgtc tactctggag atttccggtt
     6001 aaggatccac tagttctaga gcggccgctg agtgaatgtg tcttcctgtc cccaaacacg
     6061 acagcaccag gaacctactt acactgagca gagaggctgt gcctccagag cctgtgacac
     6121 gcctccactt gtatatatgg atcagaggag taaatagtgg gaagcatatt tgtcacgtgt
     6181 gtaaagattt atacagttgg aaacatgtta cctaaccagg aaaggaagac tgtttcctga
     6241 taagtggaca gccgcaagcc accatgccac cctctctgac ccaccatgta tgctggctgt
     6301 gccccagttg gactcaaggc agacaggtgt tctgccttcc ttgttaattt tgtaataatt
     6361 ggagaagata tatgtcagca cacacttaca gagcacaaac gcagtatata ggtgctggat
     6421 gtatgtaaat atattcaaat tatgtataaa tatatattat atatttacaa ggaattattt
     6481 tttgtattga ttttaaatgg atgtcctgat gcacctagaa aattggtctc tctttttttt
     6541 aaatagatat ttgctaaatg ctgttcttag agtttcttaa ttttcaccga gcagaggtgg
     6601 gaaaatactt ttgctttcag ggaaaatggt gtcacattaa tttattaacg aattggtaat
     6661 atacgaaacg attaatcatc tatagttttt ttttttttgt aatttaagtg gcatttctat
     6721 gcaggcagca cggaggacta gttaatctat tgcttggact taactggtta ttggatcctt
     6781 tgagaagaga aatatttacg atatatgact aatttggggg gaaatggtgt tttgatttat
     6841 ttgtgtttca actctgctgt ccgatgagca tgtctagaca ccctaatgcc catgtttcaa
     6901 gaaacctgtt aaactctgtc accccagggt aacaattaac cagacttccc aagacaaatg
     6961 gtaccagcat cctcatccca agatgcctta atccacttct ctggagaaca gacttccatg
     7021 ggaatgatag cagcggccgc caccgcggtg gagctccaat tcgccctata gtgagtcgta
     7081 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     7141 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     7201 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     7261 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     7321 cag
//
