LOCUS       sequence-217827-        7410 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  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 7410)
  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..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1294..1347
                     /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             1354..1731
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1753..1773
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             1789..2673
                     /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(2678..2694)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2678..2694)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3781..3800)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3782..3800)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3810..3827)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3810..3826)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3819..3841)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3967..4422)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4104..4125)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4316..4335
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4449..4553
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4554..4622,4623..5414)
                     /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(4772..4791)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5585..6173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6074..6093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6327..6344
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6461..6482
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6497..6502,6503..6520,6521..6527)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6535..6551
                     /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     6540..6562
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6559..6575
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6559..6575
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6594..6614
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6596..6614
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     6643..6659
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6644..6660
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 atactaagat gagttcaaaa taatcacccg aagacagatt tgtgtcaatt gtcacattgc
       61 accatttgag ctttctgacc ctatgttgtt tccatccctg tacacatgac actgacccaa
      121 gcacagaaaa gctttgtccc cacttctttg taatcctcag cacaggtgct cttgtggctg
      181 aatggagatg ctcattttgg ttctacattt cagtggtaga gagggccagt atacagatgg
      241 caaaatccac aatgtccaaa gcccacccat gagtttaagt tagtctgttt ccttctttga
      301 tttgctcctc tgtgagctct agaagctcaa atcccagtct accatgacta tttgcaataa
      361 atgttccact cacagcagat gggaaaggct agaacccaga ccccagcagt tctcaaacca
      421 ttgactttcc ctgtggggcc tgatgtcttg tgcacgtgct gtatgcaagt gtgtgtcatg
      481 catgttgtct ttgtagttgt ttccaagaca accagctccc tgtggaaagg aaatgcttat
      541 tgtttcacca attttcccac actatattaa tattagagta tgcagatttc aaaaccattt
      601 ttttctttag caggaattta ttaattattg tgttgggaac tagttttagc ggtagttgcg
      661 gtggtgtggt tttgtgtgta aagtccatgg cacctgagaa ggtcagagaa cactttgtgg
      721 ggctgggtct ctccggcctg tctctggtta ccagggatgt agcatgggtc acctcctcac
      781 cttaggacca cagtgctaag ctgaaataca ggagccaccc agttcttagt cctgttccag
      841 aatggacatc ctgattagaa tcctccacta ggtgttatat atctgccttt taaacttgcc
      901 actcttgttt ccattacatg agtagatgga tagatgctaa tatgaacttc gtttttaatt
      961 ttaatgttat ttttttttag tttgtctgat gtttattaag caaacttttc tatgatttaa
     1021 taccacattt ctgtgtggga ctgttcattc actgttacag ttttgttgtg agtggggatt
     1081 ctgcagcccc aaattcactc aagctgaatc atgaatgaaa agaatctgct cagatctgaa
     1141 aagtgacctt catgttcctt tcttaacaac tctctcttca gctcggttgt cttccatgga
     1201 tttcagaatg aagtttttca aagtgcagag caacccgaag ctgtagacac tgtcgacatt
     1261 atcaaagttc cagttgccat tgatatcgaa ttcgagggca gaggaagtct tctaacatgc
     1321 ggtgacgtgg aggagaatcc cggccctacc ggtatgccag agccagcgaa gtctgctccc
     1381 gccccgaaaa agggctccaa gaaggcggtg actaaggcgc agaagaaagg cggcaagaag
     1441 cgcaagcgca gccgcaagga gagctattcc atctatgtgt acaaggttct gaagcaggtc
     1501 caccctgaca ccggcatttc gtccaaggcc atgggcatca tgaattcgtt tgtgaacgac
     1561 attttcgagc gcatcgcagg tgaggcttcc cgcctggcgc attacaacaa gcgctcgacc
     1621 atcacctcca gggagatcca gacggccgtg cgcctgctgc tgcctgggga gttggccaag
     1681 cacgccgtgt ccgagggtac taaggccatc accaagtaca ccagcgctaa ggatccaccg
     1741 gtcgccacca tggaggatct gtactttcag agcgataacg atggatccga aatcggtact
     1801 ggctttccat tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat
     1861 gttggtccgc gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac
     1921 gtgtggcgca acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg
     1981 atcggtatgg gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc
     2041 ttcatggatg ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac
     2101 tggggctccg ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt
     2161 gcatttatgg agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc
     2221 gagaccttcc aggccttccg caccaccgac gtcggccgca agctgatcat cgatcagaac
     2281 gtttttatcg agggtacgct gccgatgggt gtcgtccgcc cgctgactga agtcgagatg
     2341 gaccattacc gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca
     2401 aacgagctgc caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg
     2461 gactggctgc accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg
     2521 atcccaccgg ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac
     2581 atcggcccgg gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc
     2641 gcgcgctggc tgtctactct ggagatttcc ggttaaggat ccactagttc tagagcggcc
     2701 gctgaggggc catcatccta agactgcaga agaaagaatg tcacctgacc atgctgttat
     2761 attttctctg tgtcagttca tagcgtgttc caatttgcca gcagcagtgg agagggcgtc
     2821 cctctcgctg ttcataggcc tagtcattgt tcatatagga ctcattccta ccacacactg
     2881 gggagcattc tctcaaacag gaacagctta gaatctcctg tgtgctttag atgccccaag
     2941 ttgtgtctat gagacaaaat cattggtaat gcagaaaagg gacttcaact tagttatgaa
     3001 acccataaag aagagaggga tacgatttaa aaattaaaat atgtaaataa ttaaataaat
     3061 aaatgcacgg gggaggaata aataaatgca agtggggagg gcaagactta attttgaagt
     3121 acttcattat catgcttcct ttcttttatt gtaacttata acattatttt catcaccttc
     3181 cccaaagggg cttaagtcat gggaactacg ggtttaattt gtctggtaga taaacagaaa
     3241 ggagtctaga ggcgtctatg ttttctaatg gctcctgaaa tatgacacag tgtactatgg
     3301 ttgatcttag gataagaatg tgtgttttct gtcaggagag gctaccatgg tgagtatgga
     3361 ccaggcagac acctgggaag aggataatgg ggcacaggct tgagttcatt agagctattt
     3421 tcactacact tctgccttga tgctcggtgt tcctagactc actccccgag ctctgagtta
     3481 ggagctagaa ggagacaaag cagaaatgga agggaagatg agcgaggggg cctcagggtc
     3541 tcagccttag agaagtcctg ttgcaggtgg gacaatgttt ggggctacca ctggtttcat
     3601 gtagagttct aagtttctaa ttgtctacag gccatgttta tatttaaatg tatttcagaa
     3661 aacaaaatat ttttttatag tgaatcagtt tgaaacaatc agcaaactat ctggttttgc
     3721 taagaattaa caaaggctgc tgtattgatg cggccgccac cgcggtggag ctccaattcg
     3781 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     3841 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     3901 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3961 gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4021 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     4081 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     4141 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4201 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4261 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4321 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4381 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     4441 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4501 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4561 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4621 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4681 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4741 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4801 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4861 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4921 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4981 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5041 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5101 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5161 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5221 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5281 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5341 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5401 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5461 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5521 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5581 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5641 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5701 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5761 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5821 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5881 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5941 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6001 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6061 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6121 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6181 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6241 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     6301 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     6361 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     6421 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     6481 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     6541 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     6601 accctcacta aagggaacaa aagctgggta ccgggccccc cctcgaggtc gacggtatcg
     6661 ataagcttga tatcgagtca atacctgaac tgctacctta gtgacacttt taatagggtc
     6721 atgatggatg ggaccattag gagacccttg tgtctcacat cacttggaga gtccattttc
     6781 cacaagccaa tttaaaaacc tagatttctt cacttatttt ccagagaaag attatttacc
     6841 aatgccactc ttgggtgtta ggaatgctat gttggtaaat gcaggaagcc atgaatacag
     6901 agacgatact tggttacaga tacatggata ctacagggcc catggttacc agagaatgat
     6961 tggttcagtt aaaaacagac gcaatgcccc tgattagaag aaaatatcat gaacgttttt
     7021 tgatgacatg tttaattttt ctttcaactc ctcccaactc tccccacccc attttagagt
     7081 gcagacacag aaaaaagaag actctagaaa ggtaagctct cccgggttgt atcagttgct
     7141 ttaggaacat gcagtcttta agatttcccc tggtttcttt ctttttgttc cttgtgtcct
     7201 tctctttctc cctcacccct tccttctccc cattcttttt ttatttactt atttttagtc
     7261 aaattgtttt ttatattata tattctggtc atgtttcttc ctgtctaccc gctcctctca
     7321 gattgcactg cctccctccc caccaaacaa tattttctct ctttccttca atgaggaggg
     7381 gaaaaaagaa aaagaaaacc tacaaaaaaa
//
