LOCUS       sequence-208531-        5966 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 5966)
  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 5966)
  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: pMEL15
FEATURES             Location/Qualifiers
     source          1..5966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(93..126)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(274..461)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     382..406
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(469..1041)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(1042..1385)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    complement(1440..1473)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1662..1709
                     /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      1690..2672
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        2699..2803
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2804..2872,2873..3664)
                     /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(3022..3041)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3835..4423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4324..4343
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4577..4594
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4711..4732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4747..4752,4753..4770,4771..4777)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4785..4801
                     /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     4790..4812
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4809..4825
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4809..4825
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4844..4864
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4846..4864
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4882..5150
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5171..5245
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5250..5269
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      5275..5522
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5540..5559)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5541..5559)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5569..5586)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5569..5585)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5578..5600)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5813..5832)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gttgaacatt cttaggctgg tcgaatcatt tagacacggg catcgtcctc tcgaaaggtg
       61 gcataggcca ctagtggatc tgatatcacc taataacttc gtatagcata cattatacga
      121 agttatatta agggttctcg agagctcgat tacaacaggt gttgtcctct gaggacataa
      181 aatacacacc gagattcatc aactcattgc tggagttagc atatctacaa ttgggtgaaa
      241 tggggagcga tttgcaggca tttgctcggc atgccggtag aggtgtggtc aataagagcg
      301 acctcatgct atacctgaga aagcaacctg acctacagga aagagttact caagaataag
      361 aattttcgtt ttaaaaccta agagtcactt taaaatttgt atacacttat tttttttata
      421 acttatttaa taataaaaat cataaatcat aagaaattcg cctcgagatt aggggcaggg
      481 catgctcatg tagagcgcct gctcgccgtc cgaggcggtg ccgtcgtaca gggcggtgtc
      541 caggccgcag agggtgaacc ccatccgccg gtacgcgtgg atcgccggtg cgttgacgtt
      601 ggtgacctcc agccagaggt gcccggcgcc ccgctcgcgg gcgaactccg tcgcgagccc
      661 catcaacgcg cgcccgaccc cgtgcccccg gtgctccggg gcgacctcga tgtcctcgac
      721 ggtcagccgg cggttccagc cggagtacga gacgaccacg aagcccgcca ggtcgccgtc
      781 gtccccgtac gcgacgaacg tccgggagtc cgggtcgccg tcctccccgt cgtccgattc
      841 gtcgtccgat tcgtcgtcgg ggaacacctt ggtcaggggc gggtccaccg gcacctcccg
      901 cagggtgaag ccgtccccgg tggcggtgac gcggaagacg gtgtcggtgg tgaaggaccc
      961 atccagtgcc tcgatggcct cggcgtcccc cgggacactg gtgcggtacc ggtaagccgt
     1021 gtcgtcaaga gtggtaccca tggttgttta tgttcggatg tgatgtgaga actgtatcct
     1081 agcaagattt taaaaggaag tatatgaaag aagaacctca gtggcaaatc ctaacctttt
     1141 atatttctct acaggggcgc ggcgtgggga caattcaacg cgtctgtgag gggagcgttt
     1201 ccctgctcgc aggtctgcag cgaggagccg taatttttgc ttcgcgccgt gcggccatca
     1261 aaatgtatgg atgcaaatga ttatacatgg ggatgtatgg gctaaatgta cgggcgacag
     1321 tcacatcatg cccctgagct gcgcacgtca agactgtcaa ggagggtatt ctgggcctcc
     1381 atgtcgctgg ccgggtgacc cggcggggac gaggcaagct aaacagatct ctagacctaa
     1441 taacttcgta tagcatacat tatacgaagt tatattaagg gttgtcgacc tgcagcgtac
     1501 gaaggtgcct attgatgatc tggcggaatg tctgccgtgc catagccatg ccttcacata
     1561 tagtacaggc aacacgcaga tataggtgcg acgtgaacag tgagctgtat gtgcgcagct
     1621 cgcgttgcat tttcggaagc gctcgttttc ggaaacgctt tgaagttcct attccgaagt
     1681 tcctattctc tagaaagtat aggaacttca gagcgctttt gaaaaccaaa agcgctctga
     1741 agacgcactt tcaaaaaacc aaaaacgcac cggactgtaa cgagctacta aaatattgcg
     1801 aataccgctt ccacaaacat tgctcaaaag tatctctttg ctatatatct ctgtgctata
     1861 tccctatata acctacccat ccacctttcg ctccttgaac ttgcatctaa actcgacctc
     1921 tacatttttt atgtttatct ctagtattac tctttagaca aaaaaattgt agtaagaact
     1981 attcatagag tgaatcgaaa acaatacgaa aatgtaaaca tttcctatac gtagtatata
     2041 gagacaaaat agaagaaacc gttcataatt ttctgaccaa tgaagaatca tcaacgctat
     2101 cactttctgt tcacaaagta tgcgcaatcc acatcggtat agaatataat cggggatgcc
     2161 tttatcttga aaaaatgcac ccgcagcttc gctagtaatc agtaaacgcg ggaagtggag
     2221 tcaggctttt tttatggaag agaaaataga caccaaagta gccttcttct aaccttaacg
     2281 gacctacagt gcaaaaagtt atcaagagac tgcattatag agcgcacaaa ggagaaaaaa
     2341 agtaatctaa gatgctttgt tagaaaaata gcgctctcgg gatgcatttt tgtagaacaa
     2401 aaaagaagta tagattcttt gttggtaaaa tagcgctctc gcgttgcatt tctgttctgt
     2461 aaaaatgcag ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttt
     2521 tacaaaaatg aagcacagat tcttcgttgg taaaatagcg ctttcgcgtt gcatttctgt
     2581 tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt
     2641 tgttctacaa aatgaagcac agatgcttcg ttcaggtggc acttttcggg gaaatgtgcg
     2701 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2761 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2821 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2881 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2941 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3001 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3061 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3121 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3181 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3241 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3301 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3361 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3421 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3481 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3541 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3601 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3661 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3721 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3781 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3841 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3901 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3961 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4021 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4081 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4141 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4201 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4261 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4321 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4381 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4441 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4501 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4561 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     4621 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4681 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     4741 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     4801 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
     4861 aagggaacaa aagctggagc ttctttgaaa agataatgta tgattatgct ttcactcata
     4921 tttatacaga aacttgatgt tttctttcga gtatatacaa ggtgattaca tgtacgtttg
     4981 aagtacaact ctagattttg tagtgccctc ttgggctagc ggtaaaggtg cgcatttttt
     5041 cacaccctac aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag tgaaagttgg
     5101 tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc gatacgttct
     5161 ctatggagga gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     5221 ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt tttatgtctt cgagtcatgt
     5281 aattagttat gtcacgctta cgttcacgcc ctccccccac atccgctcta accgaaaagg
     5341 aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat
     5401 taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat
     5461 gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt
     5521 gcggccggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     5581 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     5641 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     5701 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5761 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5821 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5881 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5941 tagggtgatg gttcacgtaa tgggcc
//
