LOCUS       sequence-272483-        5644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ROSA26 knock-in vector with homology-mediated end 
            joining(HMEJ)-based method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5644)
  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 5644)
  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-EGFP HMEJ donor vector
FEATURES             Location/Qualifiers
     source          1..5644
                     /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"
     misc_feature    762..1555
                     /label=Rosa26 left arm
                     /note="left homology arm from intron 1 of the mouse Rosa26 
                     gene (Soriano, 1999)"
     misc_feature    join(1574..1639,1640..1677)
                     /label=Ad SA
                     /note="adenovirus major late transcript splice acceptor 
                     (Friedrich and Soriano, 1991)"
     primer_bind     1710..1726
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1710..1726
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      1735..1744
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1741..1743,1744..1746,1747..2460)
                     /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(1741..1743,1744..1746,1747..2457)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1786..1807)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2047..2066)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2394..2415
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2483..2507)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2553..2570)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2559..2783
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3456..3476)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3456..3474)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3495..3511)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3495..3511)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3508..3530)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3519..3535
                     /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(3543..3549,3550..3567,3568..3573))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3588..3609
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3726..3743)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3897..4485)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3977..3996)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4656..5447,5448..5516))
                     /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     5279..5298
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5517..5621)
                     /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 tgggtaccgc
      661 atcgtacgcg tacgtgtttg gctcgtcgct gattggcttc ttttcctccc gccgtgtgtg
      721 aaaacacaaa tggcgtgttt tggttggcgt aaggcgcctg tcagttaacg gcagccggag
      781 tgcgcagccg ccggcagcct cgctctgccc actgggtggg gcgggaggta ggtggggtga
      841 ggcgagctgg acgtgcgggc gcggtcggcc tctggcgggg cgggggaggg gagggagggt
      901 cagcgaaagt agctcgcgcg cgagcggccg cccaccctcc ccttcctctg ggggagtcgt
      961 tttacccgcc gccggccggg cctcgtcgtc tgattggctc tcggggccca gaaaactggc
     1021 ccttgccatt ggctcgtgtt cgtgcaagtt gagtccatcc gccggccagc gggggcggcg
     1081 aggaggcgct cccaggttcc ggccctcccc tcggccccgc gccgcagagt ctggccgcgc
     1141 gcccctgcgc aacgtggcag gaagcgcgcg ctgggggcgg ggacgggcag tagggctgag
     1201 cggctgcggg gcgggtgcaa gcacgtttcc gacttgagtt gcctcaagag gggcgtgctg
     1261 agccagacct ccatcgcgca ctccggggag tggagggaag gagcgagggc tcagttgggc
     1321 tgttttggag gcaggaagca cttgctctcc caaagtcgct ctgagttgtt atcagtaagg
     1381 gagctgcagt ggagtaggcg gggagaaggc cgcacccttc tccggagggg ggaggggagt
     1441 gttgcaatac ctttctggga gttctctgct gcctcctggc ttctgaggac cgccctgggc
     1501 ctgggagaat cccttccccc tcttccctcg tgatctgcaa ctccagtctt tctagactta
     1561 agtaagggat ctgtagggcg cagtagtcca gggtttcctt gatgatgtca tacttatcct
     1621 gtcccttttt tttccacagc tcgcggttga ggacaaactc ttcgcggtct ttccagtggg
     1681 gatcgacggt atcgtagagt cgaggccgct ctagaactag tggatccacc ggtcgccacc
     1741 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1801 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1861 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1921 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1981 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2041 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2101 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2161 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2221 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2281 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2341 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2401 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2461 agcggccgct cgagatatct agacccagct ttcttgtaca aagtggttcg attcgagggg
     2521 cccccgcggg cggccgcgag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     2581 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2641 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2701 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2761 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgatcct
     2821 ctagttggcg cgcctctaga agatgggcgg gagtcttctg ggcaggctta aaggctaacc
     2881 tggtgtgtgg gcgttgtcct gcaggggaat tgaacaggtg taaaattgga gggacaagac
     2941 ttcccacaga ttttcggttt tgtcgggaag ttttttaata ggggcaaata aggaaaatgg
     3001 gaggataggt agtcatctgg ggttttatgc agcaaaacta caggttatta ttgcttgtga
     3061 tccgcctcgg agtattttcc atcgaggtag attaaagaca tgctcacccg agttttatac
     3121 tctcctgctt gagatcctta ctacagtatg aaattacagt gtcgcgagtt agactatgta
     3181 agcagaattt taatcatttt taaagagccc agtacttcat atccatttct cccgctcctt
     3241 ctgcagcctt atcaaaaggt attttagaac actcatttta gccccatttt catttattat
     3301 actggcttat ccaaccccta gacagagcat tggcattttc cctttcctga tcttagaagt
     3361 ctgatgactc atgaaaccag acagattagt tacatacacc acaaatcgag gctgccaaac
     3421 acgtacgcgt acgatgcgag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     3481 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3541 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3601 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3661 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3721 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3781 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3841 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3901 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3961 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4021 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4081 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4141 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4201 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4261 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4321 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4381 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4441 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4501 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4561 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4621 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4681 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4741 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4801 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4861 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4921 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4981 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5041 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5101 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5161 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5221 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5281 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5341 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5401 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5461 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5521 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5581 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5641 ccac
//
