LOCUS       sequence-258137-        6243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene targeting in marmoset cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6243)
  AUTHORS   Yoshimatsu S, Okahara J, Sone T, Takeda Y, Nakamura M, Sasaki E, 
            Kishi N, Shiozawa S, Okano H
  TITLE     Robust and efficient knock-in in embryonic stem cells and 
            early-stage embryos of the common marmoset using the CRISPR-Cas9 
            system.
  JOURNAL   Sci Rep. 2019 Feb 6;9(1):1528. doi: 10.1038/s41598-018-37990-w.
  PUBMED    30728412
REFERENCE   2  (bases 1 to 6243)
  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: "Sci Rep."; 
            date: "2019-02-6"; volume: "9(1):1528. doi"; pages: " 
            10.1038/s41598-018-37990-w"
COMMENT     Sequence Label: pKI-cjPLP1e2-P15L-_lox
FEATURES             Location/Qualifiers
     source          1..6243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..23
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     15..32
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     16..32
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    73..93
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    2095..2128
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3578..3598)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(3659..3675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3659..3675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3672..3694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3683..3699
                     /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(3707..3713,3714..3731,3732..3737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3752..3773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3890..3907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4061..4649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4141..4160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4820..5611,5612..5680))
                     /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     5443..5462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5681..5785)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5853..5871
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5909..5931)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6031..6050
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgcatgcctg caggtcggga
       61 tatctgaatt atcaactttg tataataaag ttgaaggtgc actccatatg acatcttaga
      121 gcatgggact agtacttgga acacagctgc tttcagagtc tgtgacttct tgtgtgcctt
      181 tcctgtttct cagcaacact ggcatggggc ctgggatacc aggtctgggg atctcaggga
      241 ctcttagcac tttaagacac atgtgttccc aggccctggt gtgttctcct agtgccagaa
      301 agatgtttca tgctttgctg actttgtata aagtctgttt gtagctgttt tgacagaatc
      361 tcagtgtaga actgaaggtg ggcacgttag ccaagctgca ctataggagg acaaaaccac
      421 catgcaaagt gtccaaaaat tattaagccc atgtttttat tatagtgagt aaagcagtct
      481 tcccattttc atttcttgtc ctatgctagc tccctcctgt ttggactgct cctctcatat
      541 gtaaactaag aagaatcaag cattctttgc aacaaataca cacgatgctc aaaaatatcc
      601 aggagcatgc aatttccaaa gtttccttca cctggaatgt tcttcaggct aaaatcctgt
      661 ctgacaatac cagcatctct ggcctgcact catcccttcc tggacctgca agtgcatcta
      721 ccctctgtta ccacttactt ggctgcctaa attgttagtt taaaatatta ggtctatcta
      781 gctaaatctt cctcaggaat ctaaagactc ccataaggca gagtctgtgt cttttctctc
      841 ttcatatccc atataacacc cactataatg ctgggcatat agtgagtatt tcataaatag
      901 ttgatgaatg actaaaataa gcaagcaaac aaacagacca gagcaataag gaaggaggga
      961 ctgatttcat gatctctctg gcttgctatt tgaattgctg aattattatt gtttgttaaa
     1021 tatatttttt agaaattctg ggaattaaag gtaaggattt tttttttctt tccttttttt
     1081 tttttttctt gagacagaat cttgctcttg tcacccaggc tggagtgcaa tggcgcaatc
     1141 ttggctcact gcaacctcca cttcctgggt tcaagagatt ttcctgcctc agccccctga
     1201 gtagctgaga ttacaggcat gtgccaccat gcctggctaa ttttcatatc tttagtagag
     1261 atggtgtttc accatgttgg ccaagctggt ctcgaactcc tgacctcagg taatcctcct
     1321 tagcctccca aagtgctggg attacaggtg tgagccacgg cgccaggcca aggatttatt
     1381 ttcaatgtaa ggattctggg tcgatctcac atgctgaaag ccaaaaccct tagtttctcc
     1441 tgcttcttga ctctggggtg cccactatct ccaagcctgt aagtgcagag cctggctggc
     1501 ctggaagaga gctttgagtg gcatgagcta cctactggat gtgcctgact gtttcccctt
     1561 cttcttcccc aggcttgtta gaatgttgcg ctcgttgctt agttggagct ctctttgctt
     1621 ccctggtggc cactggattg tgtttctttg gggtggcact gttctgtggc tgtggacatg
     1681 aagccctcac tggcacagaa aagctaattg aaacctattt ctccaaaaac tatcaagact
     1741 atgagtatct catcaatgtg taagtacttg ccctcccaca cagacccatc tttttttttt
     1801 tccctctctc catcctggag atagagtact ctaatatctt agtaactagg ggatgggtgg
     1861 agccagacag gattcctgag tcttccctct gtgcagattt ctcctgttcc acattcaaat
     1921 cctcaaatgt ggggaaattg ccagattcag aaaggagagg tctgcctgcc tgcctgcctg
     1981 ccagcctttc ccagcccctg ttttctcaca cgtgctctga cttctgctag gtgtggttaa
     2041 tattgcccaa gttggagcct ccacagtggt aggtatggag aacccaagag aggcataact
     2101 tcgtataatg tatgctatac gaagttattc taagcctctc ctgttcctag aacaacttag
     2161 gctcctgttc cttcacccac ctttcttctt acctagctcc ccactcctgt gaggtggcat
     2221 gtctgaagga tcgggcaggg agagtagggc cctggttctc caggtcacag ggtaagcaag
     2281 atgtggcaga aagatcatat gtttctggtc acatacactg tgtcttcact tatttcaaca
     2341 aagatagggt aaaagtatgc aggaggaacc tggtgattcc tctagagaat ccctagcttt
     2401 gttagggtgc ctgctctggc gtataccaca tttatgtgag taaaggagct gggtcttcaa
     2461 ttaatacgat tccttcgtct ctttcatctc cctgttactg caggatccat gccttccagt
     2521 atgtcatcta tggaactgcc tctttcttct tcctttatgg ggccctcctg ctggctgagg
     2581 gcttctacac caccggcgca gtcaggcaga tctttggcga ctacaagacc accatctgcg
     2641 gcaagggcct gagcgcaacg gtaacagggg gccagaaggg gaggggttcc agaggccaac
     2701 atcaagctca ttctttggag cgggtgtgtc attgtttggg aaaatggcta ggacatcccg
     2761 acaaggtgat catcctcagg attttgtggc aataacaagg ggtgggggaa aattgggcgt
     2821 gagtctgtgg cctcgtcccc acccaaggct gggtcctctc taggggcccg gcatttgagt
     2881 gaggaagcga tggctgcagc cgaacgagaa gatcaggaag aacgtggtgc ccagctggct
     2941 tagcctcacc tttcaaaggt tccctaagca aatttctttt caaaatggaa agcatgagtt
     3001 atgtaggatg ctttgtacaa tcagaccatt tctaagccac ctgttggtat ccctttactc
     3061 ccttcctagc agggaccaca agagtggata taactggaca agagtctaaa atactgctcg
     3121 tgagattgag acttggacac ctgatctgag aaggaggatt gataataaaa attaaataat
     3181 tgactccaag gtgaaattta ccatattccg ggatcctact ctgttggaag tcccagaggg
     3241 aattggagta gatctgcatg ttgaatgttt tatcattgtc tgagggttat ccatctgtgc
     3301 tttgagtggg gtgcacattg gttttcagta tctggagtta aagatccttt gagggctgaa
     3361 catgtgcagg ttctagccat gtctttttgt ggccagcagt cagcttagaa aggttttgta
     3421 tatgaaattt taatctccta ttggctttgt tcaatgacta gggaacaaaa atgttcccgt
     3481 ggcaaggaac attttctaag ctcagcctaa ggcacaaaac ggcacgactt tctttcaaga
     3541 tctattttga tttctttaca ccttatctgc ccagaaacaa cttttctata caaagttgat
     3601 agcttgatat caatactcta gaggatcccc gggtaccgag ctcgaattcg taatcatggt
     3661 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3721 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3781 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3841 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3901 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3961 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4021 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4081 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4141 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4201 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4261 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4321 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4381 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4441 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4501 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4561 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4621 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4681 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4741 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4801 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4861 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4921 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4981 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5041 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5101 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5161 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5221 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5281 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5341 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5401 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5461 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5521 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5581 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5641 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5701 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5761 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     5821 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     5881 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     5941 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     6001 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     6061 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
     6121 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     6181 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     6241 ttt
//
