LOCUS       sequence-208540-        6622 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  amdS 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 6622)
  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 6622)
  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: pROS11
FEATURES             Location/Qualifiers
     source          1..6622
                     /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)."
     terminator      4439..4636
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    4658..4691
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4814..5082
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5103..5177
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5182..5201
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      5426..6407
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(6453..6472)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(6477..6551)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(6572..6622,1..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 aacaaggatc catgccacaa tcttgggaag aattggctgc tgacaagaga
     2821 gctagattgg ctaagactat cccagacgaa tggaaggttc aaactttgcc agctgaagac
     2881 tctgttatcg acttcccaaa gaagtctggt atcttgtctg aagctgaatt gaagatcact
     2941 gaagcttctg ctgctgactt ggtttctaag ttggctgctg gtgaattgac ttctgttgaa
     3001 gttactttgg ctttctgtaa gagagctgct atcgctcaac aattgactaa ctgtgctcac
     3061 gaattcttcc cagacgctgc tttggctcaa gctagagaat tggacgaata ctacgctaag
     3121 cacaagagac cagttggtcc attgcacggt ttgccaatct ctttgaagga ccaattgaga
     3181 gttaagggtt acgaaacttc tatgggttac atctcttggt tgaacaagta cgacgaaggt
     3241 gactctgttt tgactactat gttgagaaag gctggtgctg ttttctacgt taagacttct
     3301 gttccacaaa ctttgatggt ttgtgaaact gttaacaaca tcatcggtag aactgttaac
     3361 ccaagaaaca agaactggtc ttgtggtggt tcttctggtg gtgaaggtgc tatcgttggt
     3421 atcagaggtg gtgttatcgg tgttggtact gacatcggtg gttctatcag agttccagct
     3481 gctttcaact tcttgtacgg tttgagacca tctcacggta gattgccata cgctaagatg
     3541 gctaactcta tggaaggtca agaaactgta cactctgttg ttggtccaat cactcactct
     3601 gttgaagact tgagattgtt cactaagtct gttttgggtc aagaaccatg gaagtacgac
     3661 tctaaggtta tcccaatgcc atggagacaa tctgaatctg acatcatcgc ttctaagatc
     3721 aagaacggtg gtttgaacat cggttactac aacttcgacg gtaacgtttt gccacaccca
     3781 ccaatcttga gaggtgttga aactactgtt gctgctttgg ctaaggctgg tcacactgtt
     3841 actccatgga ctccatacaa gcacgacttc ggtcacgact tgatctctca catctacgct
     3901 gctgacggtt ctgctgacgt tatgagagac atctctgctt ctggtgaacc agctatccca
     3961 aacatcaagg acttgttgaa cccaaacatc aaggctgtta acatgaacga attgtgggac
     4021 actcacttgc aaaagtggaa ctaccaaatg gaatacttgg aaaagtggag agaagctgaa
     4081 gaaaaggctg gtaaggaatt ggacgctatc atcgctccaa tcactccaac tgctgctgtt
     4141 agacacgacc aattcagata ctacggttac gcttctgtta tcaacttgtt ggacttcact
     4201 tctgttgttg ttccagttac tttcgctgac aagaacatcg acaagaagaa cgaatctttc
     4261 aaggctgttt ctgaattgga cgctttggtt caagaagaat acgacccaga agcttaccac
     4321 ggtgctccag ttgctgttca agttatcggt agaagattgt ctgaagaaag aactttggct
     4381 atcgctgaag aagttggtaa gttgttgggt aacgttgtta ctccataact cgagcagtac
     4441 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
     4501 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
     4561 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
     4621 gctggtcgct atactggagc tctcgagaac ccttaatata acttcgtata atgtatgcta
     4681 tacgaagtta ttaggtgata tcagatccac tagtggccta tgccaccttt cgagaggacg
     4741 atgcccgtgt ctaaatgatt cgaccagcct aagaatgttc aacccctcac taaagggaac
     4801 aaaagctgga gcttctttga aaagataatg tatgattatg ctttcactca tatttataca
     4861 gaaacttgat gttttctttc gagtatatac aaggtgatta catgtacgtt tgaagtacaa
     4921 ctctagattt tgtagtgccc tcttgggcta gcggtaaagg tgcgcatttt ttcacaccct
     4981 acaatgttct gttcaaaaga ttttggtcaa acgctgtaga agtgaaagtt ggtgcgcatg
     5041 tttcggcgtt cgaaacttct ccgcagtgaa agataaatga tcacttgaag attctttagt
     5101 gtgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     5161 gtggcaccga gtcggtggtg ctttttttgt tttttatgtc ttcgagtcat gtaattagtt
     5221 atgtcacgct tacgttcacg ccctccacgc atttaagcat aaacacgcac tatgccgttc
     5281 ttctcatgta tatatatata caggcaacac gcagatatag gtgcgacgtg aacagtgagc
     5341 tgtatgtgcg cagctcgcgt tgcattttcg gaagcgctcg ttttcggaaa cgctttgaag
     5401 ttcctattcc gaagttccta ttctgtagaa agtataggaa cctcagagcg cttttgaaaa
     5461 ccaaaagcgc tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc
     5521 tactaaaata ttgcgaatac cgcttccaca aacattgctc aaaagtatct ctttgctata
     5581 tatctctgtg ctatatccct atataaccta cccatccacc tttcgctcct tgaacttgca
     5641 tctaaactcg acctctacat tttttatgtt tatctctagt attactcttt agacaaaaaa
     5701 attgtagtaa gaactattca tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc
     5761 tatacgtagt atatagagac aaaatagaag aaaccgttca taattttctg accaatgaag
     5821 aatcatcaac gctatcactt tctgttcaca aagtatgcgc aatccacatc ggtatagaat
     5881 ataatcgggg atgcctttat cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa
     5941 acgcgggaag tggagtcagg ctttttttat ggaagagaaa atagacacca aagtagcctt
     6001 cttctaacct taacggacct acagtgcaaa aagttatcaa gagactgcat tatagagcgc
     6061 acaaaggaga aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct ctcgggatgc
     6121 atttttgtag aacaaaaaag aagtatagat tctttgttgg taaaatagcg ctctcgcgtt
     6181 gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc
     6241 gttgcatttt tgttttacaa aaatgaagca cagattcttc gttggtaaaa tagcgctttc
     6301 gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa aattagcgct
     6361 ctcgcgttgc atttttgttc tacaaaatga agcacagatg cttcgttgga gggcgtgaac
     6421 gtaagcgtga cataactaat tacatgactc gaagacataa aaaacaaaaa aagcaccacc
     6481 gactcggtgc cactttttca agttgataac ggactagcct tattttaact tgctatttct
     6541 agctctaaaa ctcctccata gagaacgtat cgatcattta tctttcactg cggagaagtt
     6601 tcgaacgccg aaacatgcgc ac
//
