LOCUS       sequence-203548-        5660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nat 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 5660)
  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 5660)
  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: pROS15
FEATURES             Location/Qualifiers
     source          1..5660
                     /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    2346..2379
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2434..2777
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2778..3353
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVISYSAWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFAGERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGERQALYMSMPCP"
     terminator      3361..3548
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3416..3440)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3696..3729
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3852..4120
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        4141..4215
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4220..4239
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     protein_bind    4435..4482
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     rep_origin      4464..5445
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(5491..5510)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5515..5589)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5610..5660,1..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 gcaccttcgt acgctgcagg tcgacaaccc
     2341 ttaatataac ttcgtataat gtatgctata cgaagttatt aggtctagag atctgtttag
     2401 cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa taccctcctt
     2461 gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc
     2521 atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc
     2581 aaaaattacg gctcctcgct gcagacctgc gagcagggaa acgctcccct cacagacgcg
     2641 ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga tttgccactg
     2701 aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc tcacatcaca
     2761 tccgaacata aacaaccatg ggtaccactc ttgacgacac ggcttaccgg taccgcacca
     2821 gtgtcccggg ggacgccgag gccatcgagg cactggatgg gtccttcacc accgacaccg
     2881 tcttccgcgt caccgccacc ggggacggct tcaccctgcg ggaggtgccg gtggacccgc
     2941 ccctgaccaa ggtgttcccc gacgacgaat cggacgacga atcggacgac ggggaggacg
     3001 gcgacccgga ctcccggacg ttcgtcgcgt acggggacga cggcgacctg gcgggcttcg
     3061 tggtcatctc gtactcggcg tggaaccgcc ggctgaccgt cgaggacatc gaggtcgccc
     3121 cggagcaccg ggggcacggg gtcgggcgcg cgttgatggg gctcgcgacg gagttcgccg
     3181 gcgagcgggg cgccgggcac ctctggctgg aggtcaccaa cgtcaacgca ccggcgatcc
     3241 acgcgtaccg gcggatgggg ttcaccctct gcggcctgga caccgccctg tacgacggca
     3301 ccgcctcgga cggcgagcgg caggcgctct acatgagcat gccctgcccc taatctcgag
     3361 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta
     3421 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttgttctt gagtaactct
     3481 ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc
     3541 tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca attgtagata
     3601 tgctaactcc agcaatgagt tgatgaatct cggtgtgtat tttatgtcct cagaggacaa
     3661 cacctgttgt aatcgagctc tcgagaaccc ttaatataac ttcgtataat gtatgctata
     3721 cgaagttatt aggtgatatc agatccacta gtggcctatg ccacctttcg agaggacgat
     3781 gcccgtgtct aaatgattcg accagcctaa gaatgttcaa cccctcacta aagggaacaa
     3841 aagctggagc ttctttgaaa agataatgta tgattatgct ttcactcata tttatacaga
     3901 aacttgatgt tttctttcga gtatatacaa ggtgattaca tgtacgtttg aagtacaact
     3961 ctagattttg tagtgccctc ttgggctagc ggtaaaggtg cgcatttttt cacaccctac
     4021 aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag tgaaagttgg tgcgcatgtt
     4081 tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc acttgaagat tctttagtgt
     4141 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     4201 ggcaccgagt cggtggtgct ttttttgttt tttatgtctt cgagtcatgt aattagttat
     4261 gtcacgctta cgttcacgcc ctccacgcat ttaagcataa acacgcacta tgccgttctt
     4321 ctcatgtata tatatataca ggcaacacgc agatataggt gcgacgtgaa cagtgagctg
     4381 tatgtgcgca gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg ctttgaagtt
     4441 cctattccga agttcctatt ctgtagaaag tataggaact tcagagcgct tttgaaaacc
     4501 aaaagcgctc tgaagacgca ctttcaaaaa accaaaaacg caccggactg taacgagcta
     4561 ctaaaatatt gcgaataccg cttccacaaa cattgctcaa aagtatctct ttgctatata
     4621 tctctgtgct atatccctat ataacctacc catccacctt tcgctccttg aacttgcatc
     4681 taaactcgac ctctacattt tttatgttta tctctagtat tactctttag acaaaaaaat
     4741 tgtagtaaga actattcata gagtgaatcg aaaacaatac gaaaatgtaa acatttccta
     4801 tacgtagtat atagagacaa aatagaagaa accgttcata attttctgac caatgaagaa
     4861 tcatcaacgc tatcactttc tgttcacaaa gtatgcgcaa tccacatcgg tatagaatat
     4921 aatcggggat gcctttatct tgaaaaaatg cacccgcagc ttcgctagta atcagtaaac
     4981 gcgggaagtg gagtcaggct ttttttatgg aagagaaaat agacaccaaa gtagccttct
     5041 tctaacctta acggacctac agtgcaaaaa gttatcaaga gactgcatta tagagcgcac
     5101 aaaggagaaa aaaagtaatc taagatgctt tgttagaaaa atagcgctct cgggatgcat
     5161 ttttgtagaa caaaaaagaa gtatagattc tttgttggta aaatagcgct ctcgcgttgc
     5221 atttctgttc tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt
     5281 tgcatttttg ttttacaaaa atgaagcaca gattcttcgt tggtaaaata gcgctttcgc
     5341 gttgcatttc tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa ttagcgctct
     5401 cgcgttgcat ttttgttcta caaaatgaag cacagatgct tcgttggagg gcgtgaacgt
     5461 aagcgtgaca taactaatta catgactcga agacataaaa aacaaaaaaa gcaccaccga
     5521 ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg ctatttctag
     5581 ctctaaaact cctccataga gaacgtatcg atcatttatc tttcactgcg gagaagtttc
     5641 gaacgccgaa acatgcgcac
//
