LOCUS       sequence-213389-        7411 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for knock-in T2A-H2B-Halo to the c-terminal of mouse 
            Dppa1 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7411)
  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 7411)
  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: Dppa1-T2A-H2B-Halo donor
FEATURES             Location/Qualifiers
     source          1..7411
                     /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             4665..4718
                     /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             4725..5102
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      5115..5124
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5124..5144
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             5160..6044
                     /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(6049..6065)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6049..6065)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(7152..7171)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7153..7171)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7181..7198)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7181..7197)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7190..7212)
                     /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 atatcgagtc
     2641 aatacctgaa ctgctacctt agtgacactt ttaatagggt catgatggat gggaccatta
     2701 ggagaccctt gtgtctcaca tcacttggag agtccatttt ccacaagcca atttaaaaac
     2761 ctagatttct tcacttattt tccagagaaa gattatttac caatgccact cttgggtgtt
     2821 aggaatgcta tgttggtaaa tgcaggaagc catgaataca gagacgatac ttggttacag
     2881 atacatggat actacagggc ccatggttac cagagaatga ttggttcagt taaaaacaga
     2941 cgcaatgccc ctgattagaa gaaaatatca tgaacgtttt ttgatgacat gtttaatttt
     3001 tctttcaact cctcccaact ctccccaccc cattttagag tgcagacaca gaaaaaagaa
     3061 gactctagaa aggtaagctc tcccgggttg tatcagttgc tttaggaaca tgcagtcttt
     3121 aagatttccc ctggtttctt tctttttgtt ccttgtgtcc ttctctttct ccctcacccc
     3181 ttccttctcc ccattctttt tttatttact tatttttagt caaattgttt tttatattat
     3241 atattctggt catgtttctt cctgtctacc cgctcctctc agattgcact gcctccctcc
     3301 ccaccaaaca atattttctc tctttccttc aatgaggagg ggaaaaaaga aaaagaaaac
     3361 ctacaaaaaa aatactaaga tgagttcaaa ataatcaccc gaagacagat ttgtgtcaat
     3421 tgtcacattg caccatttga gctttctgac cctatgttgt ttccatccct gtacacatga
     3481 cactgaccca agcacagaaa agctttgtcc ccacttcttt gtaatcctca gcacaggtgc
     3541 tcttgtggct gaatggagat gctcattttg gttctacatt tcagtggtag agagggccag
     3601 tatacagatg gcaaaatcca caatgtccaa agcccaccca tgagtttaag ttagtctgtt
     3661 tccttctttg atttgctcct ctgtgagctc tagaagctca aatcccagtc taccatgact
     3721 atttgcaata aatgttccac tcacagcaga tgggaaaggc tagaacccag accccagcag
     3781 ttctcaaacc attgactttc cctgtggggc ctgatgtctt gtgcacgtgc tgtatgcaag
     3841 tgtgtgtcat gcatgttgtc tttgtagttg tttccaagac aaccagctcc ctgtggaaag
     3901 gaaatgctta ttgtttcacc aattttccca cactatatta atattagagt atgcagattt
     3961 caaaaccatt tttttcttta gcaggaattt attaattatt gtgttgggaa ctagttttag
     4021 cggtagttgc ggtggtgtgg ttttgtgtgt aaagtccatg gcacctgaga aggtcagaga
     4081 acactttgtg gggctgggtc tctccggcct gtctctggtt accagggatg tagcatgggt
     4141 cacctcctca ccttaggacc acagtgctaa gctgaaatac aggagccacc cagttcttag
     4201 tcctgttcca gaatggacat cctgattaga atcctccact aggtgttata tatctgcctt
     4261 ttaaacttgc cactcttgtt tccattacat gagtagatgg atagatgcta atatgaactt
     4321 cgtttttaat tttaatgtta ttttttttta gtttgtctga tgtttattaa gcaaactttt
     4381 ctatgattta ataccacatt tctgtgtggg actgttcatt cactgttaca gttttgttgt
     4441 gagtggggat tctgcagccc caaattcact caagctgaat catgaatgaa aagaatctgc
     4501 tcagatctga aaagtgacct tcatgttcct ttcttaacaa ctctctcttc agctcggttg
     4561 tcttccatgg atttcagaat gaagtttttc aaagtgcaga gcaacccgaa gctgtagaca
     4621 ctgtcgacat tatcaaagtt ccagttgcca ttgatatcga attcgagggc agaggaagtc
     4681 ttctaacatg cggtgacgtg gaggagaatc ccggccctac cggtatgcca gagccagcga
     4741 agtctgctcc cgccccgaaa aagggctcca agaaggcggt gactaaggcg cagaagaaag
     4801 gcggcaagaa gcgcaagcgc agccgcaagg agagctattc catctatgtg tacaaggttc
     4861 tgaagcaggt ccaccctgac accggcattt cgtccaaggc catgggcatc atgaattcgt
     4921 ttgtgaacga cattttcgag cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca
     4981 agcgctcgac catcacctcc agggagatcc agacggccgt gcgcctgctg ctgcctgggg
     5041 agttggccaa gcacgccgtg tccgagggta ctaaggccat caccaagtac accagcgcta
     5101 aggatccacc ggtcgccacc atggaggatc tgtactttca gagcgataac gatggatccg
     5161 aaatcggtac tggctttcca ttcgaccccc attatgtgga agtcctgggc gagcgcatgc
     5221 actacgtcga tgttggtccg cgcgatggca cccctgtgct gttcctgcac ggtaacccga
     5281 cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc accgacccat cgctgcattg
     5341 ctccagacct gatcggtatg ggcaaatccg acaaaccaga cctgggttat ttcttcgacg
     5401 accacgtccg cttcatggat gccttcatcg aagccctggg tctggaagag gtcgtcctgg
     5461 tcattcacga ctggggctcc gctctgggtt tccactgggc caagcgcaat ccagagcgcg
     5521 tcaaaggtat tgcatttatg gagttcatcc gccctatccc gacctgggac gaatggccag
     5581 aatttgcccg cgagaccttc caggccttcc gcaccaccga cgtcggccgc aagctgatca
     5641 tcgatcagaa cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg
     5701 aagtcgagat ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt
     5761 ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg
     5821 aagaatacat ggactggctg caccagtccc ctgtcccgaa gctgctgttc tggggcaccc
     5881 caggcgttct gatcccaccg gccgaagccg ctcgcctggc caaaagcctg cctaactgca
     5941 aggctgtgga catcggcccg ggtctgaatc tgctgcaaga agacaacccg gacctgatcg
     6001 gcagcgagat cgcgcgctgg ctgtctactc tggagatttc cggttaagga tccactagtt
     6061 ctagagcggc cgctgagggg ccatcatcct aagactgcag aagaaagaat gtcacctgac
     6121 catgctgtta tattttctct gtgtcagttc atagcgtgtt ccaatttgcc agcagcagtg
     6181 gagagggcgt ccctctcgct gttcataggc ctagtcattg ttcatatagg actcattcct
     6241 accacacact ggggagcatt ctctcaaaca ggaacagctt agaatctcct gtgtgcttta
     6301 gatgccccaa gttgtgtcta tgagacaaaa tcattggtaa tgcagaaaag ggacttcaac
     6361 ttagttatga aacccataaa gaagagaggg atacgattta aaaattaaaa tatgtaaata
     6421 attaaataaa taaatgcacg ggggaggaat aaataaatgc aagtggggag ggcaagactt
     6481 aattttgaag tacttcatta tcatgcttcc tttcttttat tgtaacttat aacattattt
     6541 tcatcacctt ccccaaaggg gcttaagtca tgggaactac gggtttaatt tgtctggtag
     6601 ataaacagaa aggagtctag aggcgtctat gttttctaat ggctcctgaa atatgacaca
     6661 gtgtactatg gttgatctta ggataagaat gtgtgttttc tgtcaggaga ggctaccatg
     6721 gtgagtatgg accaggcaga cacctgggaa gaggataatg gggcacaggc ttgagttcat
     6781 tagagctatt ttcactacac ttctgccttg atgctcggtg ttcctagact cactccccga
     6841 gctctgagtt aggagctaga aggagacaaa gcagaaatgg aagggaagat gagcgagggg
     6901 gcctcagggt ctcagcctta gagaagtcct gttgcaggtg ggacaatgtt tggggctacc
     6961 actggtttca tgtagagttc taagtttcta attgtctaca ggccatgttt atatttaaat
     7021 gtatttcaga aaacaaaata tttttttata gtgaatcagt ttgaaacaat cagcaaacta
     7081 tctggttttg ctaagaatta acaaaggctg ctgtattgat gcggccgcca ccgcggtgga
     7141 gctccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     7201 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     7261 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     7321 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     7381 ttacgcgcag cgtgaccgct acacttgcca g
//
