LOCUS       sequence-208554-        6633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LEU2 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 6633)
  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 6633)
  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: pROS14
FEATURES             Location/Qualifiers
     source          1..6633
                     /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    4670..4703
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4825..5093
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5114..5188
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5193..5212
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      5437..6418
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(6464..6483)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(6488..6562)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(6583..6633,1..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 gcacgttcgt acgctgcagg tcgacaaccc
     2341 ttaatataac ttcgtataat gtatgctata cgaagttatt aggtctagag atccgcaggc
     2401 taaccggaac ctgtattatt tagtttatgc tacgttaaat aaagaccttt cgttcacata
     2461 actgaatgtg taatggcctt gagatttcaa gcataccaag ttggtggaga cggggtcgtt
     2521 acaaaagact ctttaagcca agatttcctt gacagccttg gcgatagcat cgccaacctc
     2581 agtggtagag ttagaaccac caaggtcacc ggttctgaca cctgcatcca agacatttct
     2641 aacagcttct tcaagagccc taccttcttc aaccaaatcc aaggataact tcaacatcat
     2701 agctgcagat aagatggtag caattgggtt aaccttgttt gctggtaaat ctggggcaga
     2761 accatgacat ggttcgtaca aaccgaatgc cttgttagtg tcaggtaggg aagctagaga
     2821 tgcagaaggt aataaaccca aagaacctgg aataacagag gcttcatcgg agataatatc
     2881 accaaacatg ttgttggtaa taacaacacc gtttagctta gttggtgatt taaccaaaat
     2941 catagcagca gagtcgatca attggtgctg aacagttaat tgtgggaact cagtcttgat
     3001 ggtttcttca acagtctttc tccacaatct ggaagaggca agcacgttag ccttgtcaag
     3061 agaccagatt ggtaatggtg ggttttgttg caatgccaag aaagcagcca ttcttgtaat
     3121 tctttgaact tcaggaacac tgtatttctc agagtcccaa gcaactccgt caccttcatc
     3181 ttcttttctt tcaccaaagt agataccacc aaccaattct ctaacgacga cgaaatcggt
     3241 accctttgca tattcaggct tcaaaggaga aagatctagt aaagaatcag aagcaaagtt
     3301 acatggtctc aagttggcgt atagacccaa ttcctttctg atcttcaata gaccttgttc
     3361 tggtctaact gcgcccgtac cccattttgg accaccaaca gcacctagta agacagcatc
     3421 ggctttctta gaggcttcta gagcttcatc tggtaaagga gtgccagtgg catcgatggc
     3481 agcacccccg atcaagtgat gttggaaatt gaacttaatt tctggacgga cttcagcaat
     3541 ggcattcaag accttaatag cttcgtcagt aacttcttta ccgacgtgat cacccggtag
     3601 gacaacgata ttcttagaca tggtataatc tgtgtagtgt gggatacttt ttacttcttc
     3661 aaataggtat caacaataaa aataaatcga agcaaatgta ggaatgcgtt aaagcagatg
     3721 tacttactta gagtacatag atatatttat ataattcaat atataaaagt atatgaagca
     3781 tctgatgttg aacctgtcat gactctaaac tggaatgggt agttatgctt ctgaaggttt
     3841 tccgttagaa aatgagtgca gggatcaggc ccggaaccgg ttttagcctg aaaggaaaag
     3901 aaaaaaatgc ggtgattact agcacgtgac tgcgctgaat tggaatctac cacaagtaca
     3961 gcgaatggca agagggaagg ggaatatatt gtgcctggcg gtagcgtctg ctatatcaaa
     4021 tgttaaacat aactaaatgc agtagtgaat agagtaatgt atttctacat atgtgggtag
     4081 ggccaatggg agcccgatgt ctattgtaca gatattttcc ttttatagtt aatcaattca
     4141 acttggaaat cattgagtcg atatctactt ggataactgg agccgtggca gcgtttgaaa
     4201 tttgacgagt cttcttgttt tgttcgcgca ttttcagttt attcaaacgg gagatggcac
     4261 cgaatccact gacttctctg ttcaatttga atggtaatag gaagaaagta catttcgaga
     4321 aattttcgcc caacgtcgaa atctcatgga tgatatcctc gatacaaatg acaccttcat
     4381 cacctaattt ctcttcaacg atattgttgt cattcaagat tatctctttt ggttctttat
     4441 cttctggcct ttgccacatt attctagatc tcttttggat taaagaacgg atagatgcaa
     4501 gtgaaggttg tccgataaca atatatggtg atacaagttt caacaatggg tacacgtcct
     4561 tggtaagttt gatgaaaaca ccagtgttca actcaacaag tctcaaaaga gataacactt
     4621 tgtaagcctt tgctgggatc ttcacagcga gctctcgaga acccttaata taacttcgta
     4681 taatgtatgc tatacgaagt tattaggtga tatcagatcc actagtggcc tatgccacct
     4741 ttcgagagga cgatgcccgt gtctaaatga tcgaccagcc taagaatgtt caacccctca
     4801 ctaaagggaa caaaagctgg agcttctttg aaaagataat gtatgattat gctttcactc
     4861 atatttatac agaaacttga tgttttcttt cgagtatata caaggtgatt acatgtacgt
     4921 ttgaagtaca actctagatt ttgtagtgcc ctcttgggct agcggtaaag gtgcgcattt
     4981 tttcacaccc tacaatgttc tgttcaaaag attttggtca aacgctgtag aagtgaaagt
     5041 tggtgcgcat gtttcggcgt tcgaaacttc tccgcagtga aagataaatg atcacttgaa
     5101 gattctttag tgtgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     5161 aacttgaaaa agtggcaccg agtcggtggt gctttttttg ttttttatgt cttcgagtca
     5221 tgtaattagt tatgtcacgc ttacgttcac gccctccacg catttaagca taaacacgca
     5281 ctatgccgtt cttctcatgt atatatatat acaggcaaca cgcagatata ggtgcgacgt
     5341 gaacagtgag ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc gttttcggaa
     5401 acgctttgaa gttcctattc cgaagttcct attctgtaga aagtatagga acctcagagc
     5461 gcttttgaaa accaaaagcg ctctgaagac gcactttcaa aaaaccaaaa acgcaccgga
     5521 ctgtaacgag ctactaaaat attgcgaata ccgcttccac aaacattgct caaaagtatc
     5581 tctttgctat atatctctgt gctatatccc tatataacct acccatccac ctttcgctcc
     5641 ttgaacttgc atctaaactc gacctctaca ttttttatgt ttatctctag tattactctt
     5701 tagacaaaaa aattgtagta agaactattc atagagtgaa tcgaaaacaa tacgaaaatg
     5761 taaacatttc ctatacgtag tatatagaga caaaatagaa gaaaccgttc ataattttct
     5821 gaccaatgaa gaatcatcaa cgctatcact ttctgttcac aaagtatgcg caatccacat
     5881 cggtatagaa tataatcggg gatgccttta tcttgaaaaa atgcacccgc agcttcgcta
     5941 gtaatcagta aacgcgggaa gtggagtcag gcttttttta tggaagagaa aatagacacc
     6001 aaagtagcct tcttctaacc ttaacggacc tacagtgcaa aaagttatca agagactgca
     6061 ttatagagcg cacaaaggag aaaaaaagta atctaagatg ctttgttaga aaaatagcgc
     6121 tctcgggatg catttttgta gaacaaaaaa gaagtataga ttctttgttg gtaaaatagc
     6181 gctctcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt
     6241 agcgctctcg cgttgcattt ttgttttaca aaaatgaagc acagattctt cgttggtaaa
     6301 atagcgcttt cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa
     6361 aaattagcgc tctcgcgttg catttttgtt ctacaaaatg aagcacagat gcttcgttgg
     6421 agggcgtgaa cgtaagcgtg acataactaa ttacatgact cgaagacata aaaaacaaaa
     6481 aaagcaccac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac
     6541 ttgctatttc tagctctaaa actcctccat agagaacgta tcgatcattt atctttcact
     6601 gcggagaagt ttcgaacgcc gaaacatgcg cac
//
