LOCUS       sequence-208528-        6942 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 6942)
  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 6942)
  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: pMEL14
FEATURES             Location/Qualifiers
     source          1..6942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    97..144
                     /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      125..1107
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        1134..1238
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1239..1307,1308..2099)
                     /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     complement(1457..1476)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2270..2858
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2759..2778
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3012..3029
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3146..3167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3182..3187,3188..3205,3206..3212)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3220..3236
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3225..3247
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3244..3260
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3244..3260
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3279..3299
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3281..3299
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3317..3585
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3606..3680
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3685..3704
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      3710..3957
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3975..3994)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3976..3994)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4004..4021)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4004..4020)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4013..4035)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4248..4267)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(4494..4527)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(6818..6851)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 acagcaacac gcagatatag gtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt
       61 tgcattttcg gaagcgctcg ttttcggaaa cgctttgaag ttcctattcc gaagttccta
      121 ttctctagaa agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc tctgaagacg
      181 cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata ttgcgaatac
      241 cgcttccaca aacattgctc aaaagtatct ctttgctata tatctctgtg ctatatccct
      301 atataaccta cccatccacc tttcgctcct tgaacttgca tctaaactcg acctctacat
      361 tttttatgtt tatctctagt attactcttt agacaaaaaa attgtagtaa gaactattca
      421 tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt atatagagac
      481 aaaatagaag aaaccgttca taattttctg accaatgaag aatcatcaac gctatcactt
      541 tctgttcaca aagtatgcgc aatccacatc ggtatagaat ataatcgggg atgcctttat
      601 cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa acgcgggaag tggagtcagg
      661 ctttttttat ggaagagaaa atagacacca aagtagcctt cttctaacct taacggacct
      721 acagtgcaaa aagttatcaa gagactgcat tatagagcgc acaaaggaga aaaaaagtaa
      781 tctaagatgc tttgttagaa aaatagcgct ctcgggatgc atttttgtag aacaaaaaag
      841 aagtatagat tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt tctgtaaaaa
      901 tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttttacaa
      961 aaatgaagca cagattcttc gttggtaaaa tagcgctttc gcgttgcatt tctgttctgt
     1021 aaaaatgcag ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttc
     1081 tacaaaatga agcacagatg cttcgttcag gtggcacttt tcggggaaat gtgcgcggaa
     1141 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1201 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1261 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1321 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1381 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1441 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1501 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1561 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1621 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1681 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1741 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     1801 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1861 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1921 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     1981 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2041 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2101 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2161 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2221 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2281 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2341 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2401 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2461 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2521 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2581 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2641 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2701 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2761 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2821 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2881 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2941 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3001 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     3061 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     3121 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     3181 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     3241 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
     3301 aacaaaagct ggagcttctt tgaaaagata atgtatgatt atgctttcac tcatatttat
     3361 acagaaactt gatgttttct ttcgagtata tacaaggtga ttacatgtac gtttgaagta
     3421 caactctaga ttttgtagtg ccctcttggg ctagcggtaa aggtgcgcat tttttcacac
     3481 cctacaatgt tctgttcaaa agattttggt caaacgctgt agaagtgaaa gttggtgcgc
     3541 atgtttcggc gttcgaaact tctccgcagt gaaagataaa tgatcgatac gttctctatg
     3601 gaggagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     3661 aaagtggcac cgagtcggtg gtgctttttt tgttttttat gtcttcgagt catgtaatta
     3721 gttatgtcac gcttacgttc acgccctccc cccacatccg ctctaaccga aaaggaagga
     3781 gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga
     3841 acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac
     3901 attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc
     3961 cggtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     4021 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4081 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     4141 gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4201 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4261 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4321 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4381 tgatggttca cgtagtgggc cgttgaacat tcttaggctg gtcgaatcat ttagacacgg
     4441 gcatcgtcct ctcgaaaggt ggcataggcc actagtggat ctgatatcac ctaataactt
     4501 cgtatagcat acattatacg aagttatatt aagggttctc gagagctcgc tgtgaagatc
     4561 ccagcaaagg cttacaaagt gttatctctt ttgagacttg ttgagttgaa cactggtgtt
     4621 ttcatcaaac ttaccaagga cgtgtaccca ttgttgaaac ttgtatcacc atatattgtt
     4681 atcggacaac cttcacttgc atctatccgt tctttaatcc aaaagagatc tagaataatg
     4741 tggcaaaggc cagaagataa agaaccaaaa gagataatct tgaatgacaa caatatcgtt
     4801 gaagagaaat taggtgatga aggtgtcatt tgtatcgagg atatcatcca tgagatttcg
     4861 acgttgggcg aaaatttctc gaaatgtact ttcttcctat taccattcaa attgaacaga
     4921 gaagtcagtg gattcggtgc catctcccgt ttgaataaac tgaaaatgcg cgaacaaaac
     4981 aagaagactc gtcaaatttc aaacgctgcc acggctccag ttatccaagt agatatcgac
     5041 tcaatgattt ccaagttgaa ttgattaact ataaaaggaa aatatctgta caatagacat
     5101 cgggctccca ttggccctac ccacatatgt agaaatacat tactctattc actactgcat
     5161 ttagttatgt ttaacatttg atatagcaga cgctaccgcc aggcacaata tattcccctt
     5221 ccctcttgcc attcgctgta cttgtggtag attccaattc agcgcagtca cgtgctagta
     5281 atcaccgcat ttttttcttt tcctttcagg ctaaaaccgg ttccgggcct gatccctgca
     5341 ctcattttct aacggaaaac cttcagaagc ataactaccc attccagttt agagtcatga
     5401 caggttcaac atcagatgct tcatatactt ttatatattg aattatataa atatatctat
     5461 gtactctaag taagtacatc tgctttaacg cattcctaca tttgcttcga tttattttta
     5521 ttgttgatac ctatttgaag aagtaaaaag tatcccacac tacacagatt ataccatgtc
     5581 taagaatatc gttgtcctac cgggtgatca cgtcggtaaa gaagttactg acgaagctat
     5641 taaggtcttg aatgccattg ctgaagtccg tccagaaatt aagttcaatt tccaacatca
     5701 cttgatcggg ggtgctgcca tcgatgccac tggcactcct ttaccagatg aagctctaga
     5761 agcctctaag aaagccgatg ctgtcttact aggtgctgtt ggtggtccaa aatggggtac
     5821 gggcgcagtt agaccagaac aaggtctatt gaagatcaga aaggaattgg gtctatacgc
     5881 caacttgaga ccatgtaact ttgcttctga ttctttacta gatctttctc ctttgaagcc
     5941 tgaatatgca aagggtaccg atttcgtcgt cgttagagaa ttggttggtg gtatctactt
     6001 tggtgaaaga aaagaagatg aaggtgacgg agttgcttgg gactctgaga aatacagtgt
     6061 tcctgaagtt caaagaatta caagaatggc tgctttcttg gcattgcaac aaaacccacc
     6121 attaccaatc tggtctcttg acaaggctaa cgtgcttgcc tcttccagat tgtggagaaa
     6181 gactgttgaa gaaaccatca agactgagtt cccacaatta actgttcagc accaattgat
     6241 cgactctgct gctatgattt tggttaaatc accaactaag ctaaacggtg ttgttattac
     6301 caacaacatg tttggtgata ttatctccga tgaagcctct gttattccag gttctttggg
     6361 tttattacct tctgcatctc tagcttccct acctgacact aacaaggcat tcggtttgta
     6421 cgaaccatgt catggttctg ccccagattt accagcaaac aaggttaacc caattgctac
     6481 catcttatct gcagctatga tgttgaagtt atccttggat ttggttgaag aaggtagggc
     6541 tcttgaagaa gctgttagaa atgtcttgga tgcaggtgtc agaaccggtg accttggtgg
     6601 ttctaactct accactgagg ttggcgatgc tatcgccaag gctgtcaagg aaatcttggc
     6661 ttaaagagtc ttttgtaacg accccgtctc caccaacttg gtatgcttga aatctcaagg
     6721 ccattacaca ttcagttatg tgaacgaaag gtctttattt aacgtagcat aaactaaata
     6781 atacaggttc cggttagcct gcggatctct agacctaata acttcgtata gcatacatta
     6841 tacgaagtta tattaagggt tgtcgacctg cagcgtacga aggtgcctat tgatgatctg
     6901 gcggaatgtc tgccgtgcca tagccatgcc ttcacatata gt
//
