LOCUS       sequence-272479-        6206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  R0SA26 knock-in vector with homologous recombination-based method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6206)
  AUTHORS   Abe T, Inoue KI, Furuta Y, Kiyonari H
  TITLE     Pronuclear Microinjection during S-Phase Increases the Efficiency of
            CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes.
  JOURNAL   Cell Rep. 2020 May 19;31(7):107653. doi: 
            10.1016/j.celrep.2020.107653.
  PUBMED    32433962
REFERENCE   2  (bases 1 to 6206)
  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: "Cell Rep.";
            date: "2020-05-19"; volume: "31(7):107653. doi"; pages: " 
            10.1016/j.celrep.2020.107653"
COMMENT     Sequence Label: R26-H2B-EGFP HR donor vector
FEATURES             Location/Qualifiers
     source          1..6206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    781..1574
                     /label=Rosa26 left arm
                     /note="left homology arm from intron 1 of the mouse Rosa26 
                     gene (Soriano, 1999)"
     misc_feature    join(1593..1658,1659..1696)
                     /label=Ad SA
                     /note="adenovirus major late transcript splice acceptor 
                     (Friedrich and Soriano, 1991)"
     primer_bind     1729..1745
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1729..1745
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    1759..1792
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1827..1851
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1900..2277
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      2290..2299
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2296..2298,2299..2301,2302..3015)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2296..2298,2299..2301,2302..3012)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2341..2362)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2602..2621)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2949..2970
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3038..3062)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3108..3125)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3114..3338
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4018..4038)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4018..4036)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4057..4073)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4057..4073)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4070..4092)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4081..4097
                     /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(4105..4111,4112..4129,4130..4135))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4150..4171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4288..4305)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4459..5047)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4539..4558)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5218..6009,6010..6078))
                     /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     5841..5860
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6079..6183)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc ctcgtcgctg attggcttct
      721 tttcctcccg ccgtgtgtga aaacacaaat ggcgtgtttt ggttggcgta aggcgcctgt
      781 cagttaacgg cagccggagt gcgcagccgc cggcagcctc gctctgccca ctgggtgggg
      841 cgggaggtag gtggggtgag gcgagctgga cgtgcgggcg cggtcggcct ctggcggggc
      901 gggggagggg agggagggtc agcgaaagta gctcgcgcgc gagcggccgc ccaccctccc
      961 cttcctctgg gggagtcgtt ttacccgccg ccggccgggc ctcgtcgtct gattggctct
     1021 cggggcccag aaaactggcc cttgccattg gctcgtgttc gtgcaagttg agtccatccg
     1081 ccggccagcg ggggcggcga ggaggcgctc ccaggttccg gccctcccct cggccccgcg
     1141 ccgcagagtc tggccgcgcg cccctgcgca acgtggcagg aagcgcgcgc tgggggcggg
     1201 gacgggcagt agggctgagc ggctgcgggg cgggtgcaag cacgtttccg acttgagttg
     1261 cctcaagagg ggcgtgctga gccagacctc catcgcgcac tccggggagt ggagggaagg
     1321 agcgagggct cagttgggct gttttggagg caggaagcac ttgctctccc aaagtcgctc
     1381 tgagttgtta tcagtaaggg agctgcagtg gagtaggcgg ggagaaggcc gcacccttct
     1441 ccggaggggg gaggggagtg ttgcaatacc tttctgggag ttctctgctg cctcctggct
     1501 tctgaggacc gccctgggcc tgggagaatc ccttccccct cttccctcgt gatctgcaac
     1561 tccagtcttt ctagacttaa gtaagggatc tgtagggcgc agtagtccag ggtttccttg
     1621 atgatgtcat acttatcctg tccctttttt ttccacagct cgcggttgag gacaaactct
     1681 tcgcggtctt tccagtgggg atcgacggta tcgtagagtc gaggccgctc tagaactagt
     1741 ggatccggaa cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtccc
     1801 tcgacctgca gcccaagcta gatcaaacaa gtttgtacaa aaaagcaggc tggcgccgga
     1861 acccaagctt cgaattctgc agtcgacggt accgccacca tgccagagcc agcgaagtct
     1921 gctcccgccc cgaaaaaggg ctccaagaag gcggtgacta aggcgcagaa gaaagacggc
     1981 aagaagcgca agcgcagccg caaggagagc tattccatct atgtgtacaa ggttctgaag
     2041 caggtccacc ctgacaccgg catttcgtcc aaggccatgg gcatcatgaa ttcgtttgtg
     2101 aacgacattt tcgagcgcat cgcaggtgag gcttcacgcc tggcgcatta caacaagcgc
     2161 tcgaccatca cctccaggga gatccagacg gccgtgcgcc tgctgctgcc tggggagttg
     2221 gccaagcacg ccgtgtccga gggtactaag gccgtcacca agtacaccag cgctaaggat
     2281 ccaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     2341 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     2401 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     2461 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     2521 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2581 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2641 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2701 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2761 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2821 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2881 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     2941 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3001 gagctgtaca agtaaagcgg ccgctcgaga tatctagacc cagctttctt gtacaaagtg
     3061 gttcgattcg aggggccccc gcgggcggcc gcgagctcgc tgatcagcct cgactgtgcc
     3121 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3181 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3241 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3301 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3361 ctggggctcg atcctctagt tggcgcgcct ctagaagatg ggcgggagtc ttctgggcag
     3421 gcttaaaggc taacctggtg tgtgggcgtt gtcctgcagg ggaattgaac aggtgtaaaa
     3481 ttggagggac aagacttccc acagattttc ggttttgtcg ggaagttttt taataggggc
     3541 aaataaggaa aatgggagga taggtagtca tctggggttt tatgcagcaa aactacaggt
     3601 tattattgct tgtgatccgc ctcggagtat tttccatcga ggtagattaa agacatgctc
     3661 acccgagttt tatactctcc tgcttgagat ccttactaca gtatgaaatt acagtgtcgc
     3721 gagttagact atgtaagcag aattttaatc atttttaaag agcccagtac ttcatatcca
     3781 tttctcccgc tccttctgca gccttatcaa aaggtatttt agaacactca ttttagcccc
     3841 attttcattt attatactgg cttatccaac ccctagacag agcattggca ttttcccttt
     3901 cctgatctta gaagtctgat gactcatgaa accagacaga ttagttacat acaccacaaa
     3961 tcgaggctgg ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct
     4021 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4081 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4141 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4201 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4261 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4321 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4381 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4441 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4501 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4561 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4621 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4681 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4741 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4801 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4861 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4921 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4981 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5041 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5101 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5161 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5221 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5281 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5341 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5401 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5461 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5521 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5581 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5641 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5701 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5761 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5821 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5881 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5941 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6001 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6061 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6121 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6181 gcgcacattt ccccgaaaag tgccac
//
