LOCUS       sequence-272482-        6006 bp    DNA     circular SYN 14-APR-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 6006)
  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 6006)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-mCherry HR donor vector
FEATURES             Location/Qualifiers
     source          1..6006
                     /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)"
     CDS             1778..2155
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      2168..2177
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2174..2884
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2319..2337)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2587..2606
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2908..2925)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2914..3138
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3818..3838)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3818..3836)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3857..3873)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3857..3873)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3870..3892)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3881..3897
                     /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(3905..3911,3912..3929,3930..3935))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3950..3971
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4088..4105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4259..4847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4339..4358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5018..5809,5810..5878))
                     /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     5641..5660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5879..5983)
                     /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 gttagcttcg aattctgcag tcgacggtac cgccaccatg ccagagccag cgaagtctgc
     1801 tcccgccccg aaaaagggct ccaagaaggc ggtgactaag gcgcagaaga aagacggcaa
     1861 gaagcgcaag cgcagccgca aggagagcta ttccatctat gtgtacaagg ttctgaagca
     1921 ggtccaccct gacaccggca tttcgtccaa ggccatgggc atcatgaatt cgtttgtgaa
     1981 cgacattttc gagcgcatcg caggtgaggc ttcacgcctg gcgcattaca acaagcgctc
     2041 gaccatcacc tccagggaga tccagacggc cgtgcgcctg ctgctgcctg gggagttggc
     2101 caagcacgcc gtgtccgagg gtactaaggc cgtcaccaag tacaccagcg ctaaggatcc
     2161 accggtcgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     2221 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     2281 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     2341 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     2401 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     2461 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     2521 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     2581 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     2641 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     2701 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     2761 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     2821 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     2881 gtaagcggcc aaacgctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2941 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3001 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3061 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3121 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg atcctctagt
     3181 tggcgcgcct ctagaagatg ggcgggagtc ttctgggcag gcttaaaggc taacctggtg
     3241 tgtgggcgtt gtcctgcagg ggaattgaac aggtgtaaaa ttggagggac aagacttccc
     3301 acagattttc ggttttgtcg ggaagttttt taataggggc aaataaggaa aatgggagga
     3361 taggtagtca tctggggttt tatgcagcaa aactacaggt tattattgct tgtgatccgc
     3421 ctcggagtat tttccatcga ggtagattaa agacatgctc acccgagttt tatactctcc
     3481 tgcttgagat ccttactaca gtatgaaatt acagtgtcgc gagttagact atgtaagcag
     3541 aattttaatc atttttaaag agcccagtac ttcatatcca tttctcccgc tccttctgca
     3601 gccttatcaa aaggtatttt agaacactca ttttagcccc attttcattt attatactgg
     3661 cttatccaac ccctagacag agcattggca ttttcccttt cctgatctta gaagtctgat
     3721 gactcatgaa accagacaga ttagttacat acaccacaaa tcgaggctgg ctagttctag
     3781 agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc
     3841 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3901 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3961 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4021 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4081 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4141 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4201 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4261 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4321 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4381 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4441 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4501 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4561 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4621 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4681 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4741 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4801 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4861 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4921 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4981 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5041 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5101 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5161 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5221 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5281 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5341 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5401 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5461 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5521 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5581 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5641 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5701 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5761 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5821 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5881 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5941 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6001 tgccac
//
