LOCUS       sequence-208541-        5809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  kan based Yeast/E.coli shuttle vector designed to clone and express 
            Cas9 programming spacer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5809)
  AUTHORS   Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NG, van den 
            Broek M, Daran-Lapujade P, Pronk JT, van Maris AJ, Daran JM
  TITLE     CRISPR/Cas9: a molecular Swiss army knife for simultaneous 
            introduction of multiple genetic modifications in Saccharomyces 
            cerevisiae.
  JOURNAL   FEMS Yeast Res. 2015 Mar;15(2). pii: fov004. doi: 
            10.1093/femsyr/fov004. Epub 2015 Mar 4.
  PUBMED    25743786
REFERENCE   2  (bases 1 to 5809)
  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: "FEMS Yeast 
            Res."; date: "2015-03"; volume: "15(2). pii: fov004. doi"; pages: " 
            10.1093/femsyr/fov004. Epub 2015 Mar 4"
COMMENT     Sequence Label: pROS13
FEATURES             Location/Qualifiers
     source          1..5809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(351..368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(522..1110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(602..621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1281..2072,2073..2141))
                     /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     1904..1923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2142..2246)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    2338..2371
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            2426..3782
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2426..2769
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2770..3579
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2837..2856)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3585..3782
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    3845..3878
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4001..4269
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        4290..4364
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4369..4388
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4613..5594
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(5640..5659)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5664..5738)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5759..5809,1..218))
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
ORIGIN
        1 caactttcac ttctacagcg tttgaccaaa atcttttgaa cagaacattg tagggtgtga
       61 aaaaatgcgc acctttaccg ctagcccaag agggcactac aaaatctaga gttgtacttc
      121 aaacgtacat gtaatcacct tgtatatact cgaaagaaaa catcaagttt ctgtataaat
      181 atgagtgaaa gcataatcat acattatctt ttcaaagaag ctccagcttt tgttcccttt
      241 agtgagggta ttcacgtaga cggataggta tagccagaca tcagcagcat acttcgggaa
      301 ccgtaggctc tagagagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
      361 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      421 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      481 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      541 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      601 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      661 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      721 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      781 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      841 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      901 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      961 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1021 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1081 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1141 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1201 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     1261 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1321 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1381 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1441 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1501 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1561 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1621 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1681 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1741 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1801 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1861 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1921 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1981 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     2041 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     2101 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     2161 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2221 aaaaataaac aaataggggt tccgcgcact cgagactata tgtgaaggca tggctatggc
     2281 acggcagaca ttccgccaga tcatcaatag gcacgcggcc ggtcgacaac ccttaatata
     2341 acttcgtata atgtatgcta tacgaagtta ttaggtctag agatctgttt agcttgcctc
     2401 gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct
     2461 tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc
     2521 ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta
     2581 cggctcctcg ctgcagacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg
     2641 tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt
     2701 ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca
     2761 taaacaacca tgggtaagga aaagactcac gtttcgaggc cgcgattaaa ttccaacatg
     2821 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     2881 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     2941 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     3001 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     3061 gcgatccccg gcaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     3121 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     3181 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     3241 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     3301 aaagaaatgc ataagctttt gccattctca ccggattcag tcgtcactca tggtgatttc
     3361 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     3421 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     3481 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     3541 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagtactgac aataaaaaga
     3601 ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt
     3661 aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga
     3721 agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac
     3781 tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa aacgagctct cgagaaccct
     3841 taatataact tcgtataatg tatgctatac gaagttatta ggtgatatca gatccactag
     3901 tggcctatgc cacctttcga gaggacgatg cccgtgtcta aatgattcga ccagcctaag
     3961 aatgttcaac ccctcactaa agggaacaaa agctggagct tctttgaaaa gataatgtat
     4021 gattatgctt tcactcatat ttatacagaa acttgatgtt ttctttcgag tatatacaag
     4081 gtgattacat gtacgtttga agtacaactc tagattttgt agtgccctct tgggctagcg
     4141 gtaaaggtgc gcattttttc acaccctaca atgttctgtt caaaagattt tggtcaaacg
     4201 ctgtagaagt gaaagttggt gcgcatgttt cggcgttcga aacttctccg cagtgaaaga
     4261 taaatgatca cttgaagatt ctttagtgtg ttttagagct agaaatagca agttaaaata
     4321 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtggtgctt tttttgtttt
     4381 ttatgtcttc gagtcatgta attagttatg tcacgcttac gttcacgccc tccacgcatt
     4441 taagcataaa cacgcactat gccgttcttc tcatgtatat atatatacag gcaacacgca
     4501 gatataggtg cgacgtgaac agtgagctgt atgtgcgcag ctcgcgttgc attttcggaa
     4561 gcgctcgttt tcggaaacgc tttgaagttc ctattccgaa gttcctattc tgtagaaagt
     4621 ataggaacct cagagcgctt ttgaaaacca aaagcgctct gaagacgcac tttcaaaaaa
     4681 ccaaaaacgc accggactgt aacgagctac taaaatattg cgaataccgc ttccacaaac
     4741 attgctcaaa agtatctctt tgctatatat ctctgtgcta tatccctata taacctaccc
     4801 atccaccttt cgctccttga acttgcatct aaactcgacc tctacatttt ttatgtttat
     4861 ctctagtatt actctttaga caaaaaaatt gtagtaagaa ctattcatag agtgaatcga
     4921 aaacaatacg aaaatgtaaa catttcctat acgtagtata tagagacaaa atagaagaaa
     4981 ccgttcataa ttttctgacc aatgaagaat catcaacgct atcactttct gttcacaaag
     5041 tatgcgcaat ccacatcggt atagaatata atcggggatg cctttatctt gaaaaaatgc
     5101 acccgcagct tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt tttttatgga
     5161 agagaaaata gacaccaaag tagccttctt ctaaccttaa cggacctaca gtgcaaaaag
     5221 ttatcaagag actgcattat agagcgcaca aaggagaaaa aaagtaatct aagatgcttt
     5281 gttagaaaaa tagcgctctc gggatgcatt tttgtagaac aaaaaagaag tatagattct
     5341 ttgttggtaa aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc agctcagatt
     5401 ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa tgaagcacag
     5461 attcttcgtt ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa aatgcagctc
     5521 agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttctac aaaatgaagc
     5581 acagatgctt cgttggaggg cgtgaacgta agcgtgacat aactaattac atgactcgaa
     5641 gacataaaaa acaaaaaaag caccaccgac tcggtgccac tttttcaagt tgataacgga
     5701 ctagccttat tttaacttgc tatttctagc tctaaaactc ctccatagag aacgtatcga
     5761 tcatttatct ttcactgcgg agaagtttcg aacgccgaaa catgcgcac
//
