LOCUS       sequence-203544-        6615 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 6615)
  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 6615)
  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..6615
                     /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)."
     protein_bind    4651..4684
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4807..5075
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5096..5170
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5175..5194
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     protein_bind    5390..5437
                     /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      5419..6400
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(6446..6465)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(6470..6544)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(6565..6615,1..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 cgaggacaat
     4441 aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt
     4501 ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca
     4561 gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc
     4621 gctatactgg agctctcgag aacccttaat ataacttcgt ataatgtatg ctatacgaag
     4681 ttattaggtg atatcagatc cactagtggc ctatgccacc tttcgagagg acgatgcccg
     4741 tgtctaaatg attcgaccag cctaagaatg ttcaacccct cactaaaggg aacaaaagct
     4801 ggagcttctt tgaaaagata atgtatgatt atgctttcac tcatatttat acagaaactt
     4861 gatgttttct ttcgagtata tacaaggtga ttacatgtac gtttgaagta caactctaga
     4921 ttttgtagtg ccctcttggg ctagcggtaa aggtgcgcat tttttcacac cctacaatgt
     4981 tctgttcaaa agattttggt caaacgctgt agaagtgaaa gttggtgcgc atgtttcggc
     5041 gttcgaaact tctccgcagt gaaagataaa tgatcacttg aagattcttt agtgtgtttt
     5101 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     5161 cgagtcggtg gtgctttttt tgttttttat gtcttcgagt catgtaatta gttatgtcac
     5221 gcttacgttc acgccctcca cgcatttaag cataaacacg cactatgccg ttcttctcat
     5281 gtatatatat atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt
     5341 gcgcagctcg cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat
     5401 tccgaagttc ctattctgta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag
     5461 cgctctgaag acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa
     5521 atattgcgaa taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct
     5581 gtgctatatc cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac
     5641 tcgacctcta cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag
     5701 taagaactat tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt
     5761 agtatataga gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc
     5821 aacgctatca ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg
     5881 gggatgcctt tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg
     5941 aagtggagtc aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa
     6001 ccttaacgga cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg
     6061 agaaaaaaag taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg
     6121 tagaacaaaa aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc
     6181 tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat
     6241 ttttgtttta caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc
     6301 atttctgttc tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt
     6361 tgcatttttg ttctacaaaa tgaagcacag atgcttcgtt ggagggcgtg aacgtaagcg
     6421 tgacataact aattacatga ctcgaagaca taaaaaacaa aaaaagcacc accgactcgg
     6481 tgccactttt tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta
     6541 aaactcctcc atagagaacg tatcgatcat ttatctttca ctgcggagaa gtttcgaacg
     6601 ccgaaacatg cgcac
//
