LOCUS       sequence-217896-        7322 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7322)
  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 7322)
  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..7322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..21
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             37..921
                     /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(926..942)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(926..942)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1986..2005)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1987..2005)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2015..2032)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2015..2031)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2024..2046)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2172..2627)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2309..2330)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2521..2540
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2654..2758
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2759..2827,2828..3619)
                     /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(2977..2996)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3790..4378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4279..4298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4532..4549
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4666..4687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4702..4707,4708..4725,4726..4732)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4740..4756
                     /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     4745..4767
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4764..4780
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4764..4780
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4799..4819
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4801..4819
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4848..4864
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4849..4865
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             6864..6917
                     /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             6924..7301
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      join(7314..7322,1)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 gaggatctgt actttcagag cgataacgat ggatccgaaa tcggtactgg ctttccattc
       61 gacccccatt atgtggaagt cctgggcgag cgcatgcact acgtcgatgt tggtccgcgc
      121 gatggcaccc ctgtgctgtt cctgcacggt aacccgacct cctcctacgt gtggcgcaac
      181 atcatcccgc atgttgcacc gacccatcgc tgcattgctc cagacctgat cggtatgggc
      241 aaatccgaca aaccagacct gggttatttc ttcgacgacc acgtccgctt catggatgcc
      301 ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca ttcacgactg gggctccgct
      361 ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca aaggtattgc atttatggag
      421 ttcatccgcc ctatcccgac ctgggacgaa tggccagaat ttgcccgcga gaccttccag
      481 gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt ttttatcgag
      541 ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga ccattaccgc
      601 gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca
      661 atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga ctggctgcac
      721 cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat cccaccggcc
      781 gaagccgctc gcctggccaa aagcctgcct aactgcaagg ctgtggacat cggcccgggt
      841 ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc gcgctggctg
      901 tctactctgg agatttccgg ttaaggatcc actagttcta gagcggccgc tgagtgaatg
      961 tgtcttcctg tccccaaaca cgacagcacc aggaacctac ttacactgag cagagaggct
     1021 gtgcctccag agcctgtgac acgcctccac ttgtatatat ggatcagagg agtaaatagt
     1081 gggaagcata tttgtcacgt gtgtaaagat ttatacagtt ggaaacatgt tacctaacca
     1141 ggaaaggaag actgtttcct gataagtgga cagccgcaag ccaccatgcc accctctctg
     1201 acccaccatg tatgctggct gtgccccagt tggactcaag gcagacaggt gttctgcctt
     1261 ccttgttaat tttgtaataa ttggagaaga tatatgtcag cacacactta cagagcacaa
     1321 acgcagtata taggtgctgg atgtatgtaa atatattcaa attatgtata aatatatatt
     1381 atatatttac aaggaattat tttttgtatt gattttaaat ggatgtcctg atgcacctag
     1441 aaaattggtc tctctttttt ttaaatagat atttgctaaa tgctgttctt agagtttctt
     1501 aattttcacc gagcagaggt gggaaaatac ttttgctttc agggaaaatg gtgtcacatt
     1561 aatttattaa cgaattggta atatacgaaa cgattaatca tctatagttt tttttttttt
     1621 gtaatttaag tggcatttct atgcaggcag cacggaggac tagttaatct attgcttgga
     1681 cttaactggt tattggatcc tttgagaaga gaaatattta cgatatatga ctaatttggg
     1741 gggaaatggt gttttgattt atttgtgttt caactctgct gtccgatgag catgtctaga
     1801 caccctaatg cccatgtttc aagaaacctg ttaaactctg tcaccccagg gtaacaatta
     1861 accagacttc ccaagacaaa tggtaccagc atcctcatcc caagatgcct taatccactt
     1921 ctctggagaa cagacttcca tgggaatgat agcagcggcc gccaccgcgg tggagctcca
     1981 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     2041 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     2101 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2161 atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2221 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2281 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2341 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2401 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2461 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2521 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2581 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     2641 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2701 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2761 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2821 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2881 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2941 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3001 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3061 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3121 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3181 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3241 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3301 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3361 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3421 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3481 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3541 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3601 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3661 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3721 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3781 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3841 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3901 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     3961 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4021 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4081 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4141 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4201 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4261 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4321 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4381 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4441 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4501 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4561 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     4621 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     4681 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     4741 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
     4801 aattaaccct cactaaaggg aacaaaagct gggtaccggg ccccccctcg aggtcgacgg
     4861 tatcgataag cttgatatct gctgatgttg ttggagccga tacatccccc ttcttggatg
     4921 gcgactcttt ctcttgccaa ccctagtgat gaattatagt gggtttttgt cttttaaacc
     4981 ctatgatcgc caacattcca tgggctctct gctgataatg ctgaggctgt ttgtgctgta
     5041 gtctgcgctt tttgccccct ctcagaaaaa ctgtatgtca taggagttgc tggctattgg
     5101 gtacataagc aaagccaccc tattgtgcca gtgccttaga cagtgagaca agaaaggccc
     5161 ctggttagaa atcttatcag gactgggaat gtaactcagt tgataagagt gcttgcttag
     5221 cgtgcacaca gccctgggtt caaccgccta gtactacaga aactgagtgt ggcttcacac
     5281 acctgtaatc ccagcacttg gagagataga tgcaggagga ttagaagttc aaggttatct
     5341 ttagtcacat agtattggta gccagccagc ctggaatact tgagatactt acaggaagga
     5401 aggaaggaag gaaagaagga gggagagagg acaggaggaa ggagatagat atacacagaa
     5461 agagacagag aaacagagat tcaggagaca caaagacata cggagacaca gtgagagaaa
     5521 cagaaagaca cagagagaga cagagagaca taaagagaaa tcccttctag ttttgtccag
     5581 ggtcagttag taagtacttt ctgtaaggcc caaagagtaa acattttcag cttcaaggcc
     5641 atatggtctc tgtcgaagct gccattctag catgaaagca gccacagctc tatgtaaata
     5701 aagagggtct gtgttccact aaactttatg gactctgaaa ctgggaaatt tatataattt
     5761 catataaatt tatatcattt catgtcattg gctgccaaaa gatatgactc aatttgattt
     5821 ttctactagc catgtacaaa tgttgagaag tcgccttagt tcatggacca aacagaaaca
     5881 cacatgggtt tgagccttgt cctgagctgg aggaagagag tgacccaaag ggaccttggt
     5941 agcagccagg gatgtgttgg ggagcagaga aacttttatg aacttcagtt tcagtactga
     6001 aacttccctt tccctagact tcctttgaca gcccattacg ggcttaacat ccatgagggt
     6061 agccacacct ttccctaacc cgtttctgtt gcttactctg gagctcctga gggtaatgga
     6121 ttatatctgt taaactgtgc aacaaacgga gatgacttct cccctaaatg atcctagaaa
     6181 tgacaactgt agaatgtgat tccgaaaata catattcatg tcctggcttt gtaaagggtc
     6241 ctgctgggtt tacttcactg ggtcttaaac tccgattgtg agccgtagtg aagagggctg
     6301 tatatagtgg gtcaccgagg tcctgtagca gagtgggcaa gctcactgcc tgctaccagc
     6361 agttcactat gttttatggt ctgctgcctg ctggtggttt atagatgctg tgtcgtaaga
     6421 gaaaagttca gggtagcctg gagtgaatgg agttggggta tcagggaggt ctttgtacac
     6481 tggggtgagc taggcctctg gaaagcttcc tgggggttcc ccgtcccttc ccaaatctga
     6541 tagagatttt tcaggagctt ctgtttaaaa tgagattctt ctcttccctt cttgcaggaa
     6601 tacttggacc tcagtcagcc tctcgaaccg tattcacctt gttatcctga cccaaggtga
     6661 aataaaacgt ctctcttccc ttcttgcagg aatacttgga tctcacccag cctctcgaac
     6721 agtattctcc tagttacccc gacacaagga gctcttgttc ttcaggggac gattctgtgt
     6781 tttctccaga ccccatgcct tatgaaccct gtctgcctca gtatccacac ataaacggca
     6841 gtgttaaaac agatatcgaa ttcgagggca gaggaagtct tctaacatgc ggtgacgtgg
     6901 aggagaatcc cggccctacc ggtatgccag agccagcgaa gtctgctccc gccccgaaaa
     6961 agggctccaa gaaggcggtg actaaggcgc agaagaaagg cggcaagaag cgcaagcgca
     7021 gccgcaagga gagctattcc atctatgtgt acaaggttct gaagcaggtc caccctgaca
     7081 ccggcatttc gtccaaggcc atgggcatca tgaattcgtt tgtgaacgac attttcgagc
     7141 gcatcgcagg tgaggcttcc cgcctggcgc attacaacaa gcgctcgacc atcacctcca
     7201 gggagatcca gacggccgtg cgcctgctgc tgcctgggga gttggccaag cacgccgtgt
     7261 ccgagggtac taaggccatc accaagtaca ccagcgctaa ggatccaccg gtcgccacca
     7321 tg
//
