LOCUS       sequence-208527-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  kan 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 6126)
  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 6126)
  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: pMEL13
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    98..145
                     /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      126..1108
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        1135..1239
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1240..1308,1309..2100)
                     /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(1458..1477)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2271..2859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2760..2779
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3013..3030
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3147..3168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3183..3188,3189..3206,3207..3213)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3221..3237
                     /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     3226..3248
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3245..3261
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3245..3261
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3280..3300
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3282..3300
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3318..3586
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3607..3681
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3686..3705
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      3711..3958
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3976..3995)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3977..3995)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4005..4022)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4005..4021)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4014..4036)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4249..4268)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(4495..4528)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            complement(4591..5947)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(4591..4788)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4794..5603)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5517..5536
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5604..5947)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    complement(6002..6035)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 acaggcaaca cgcagatata ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgcg
       61 ttgcattttc ggaagcgctc gttttcggaa acgctttgaa gttcctattc cgaagttcct
      121 attctctaga aagtatagga acttcagagc gcttttgaaa accaaaagcg ctctgaagac
      181 gcactttcaa aaaaccaaaa acgcaccgga ctgtaacgag ctactaaaat attgcgaata
      241 ccgcttccac aaacattgct caaaagtatc tctttgctat atatctctgt gctatatccc
      301 tatataacct acccatccac ctttcgctcc ttgaacttgc atctaaactc gacctctaca
      361 ttttttatgt ttatctctag tattactctt tagacaaaaa aattgtagta agaactattc
      421 atagagtgaa tcgaaaacaa tacgaaaatg taaacatttc ctatacgtag tatatagaga
      481 caaaatagaa gaaaccgttc ataattttct gaccaatgaa gaatcatcaa cgctatcact
      541 ttctgttcac aaagtatgcg caatccacat cggtatagaa tataatcggg gatgccttta
      601 tcttgaaaaa atgcacccgc agcttcgcta gtaatcagta aacgcgggaa gtggagtcag
      661 gcttttttta tggaagagaa aatagacacc aaagtagcct tcttctaacc ttaacggacc
      721 tacagtgcaa aaagttatca agagactgca ttatagagcg cacaaaggag aaaaaaagta
      781 atctaagatg ctttgttaga aaaatagcgc tctcgggatg catttttgta gaacaaaaaa
      841 gaagtataga ttctttgttg gtaaaatagc gctctcgcgt tgcatttctg ttctgtaaaa
      901 atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttttaca
      961 aaaatgaagc acagattctt cgttggtaaa atagcgcttt cgcgttgcat ttctgttctg
     1021 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
     1081 ctacaaaatg aagcacagat gcttcgttca ggtggcactt ttcggggaaa tgtgcgcgga
     1141 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1201 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     1261 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1321 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     1381 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     1441 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     1501 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     1561 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     1621 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     1681 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     1741 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     1801 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     1861 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     1921 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     1981 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2041 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2101 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2161 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     2221 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2281 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2341 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2401 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     2461 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2521 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2581 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2641 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2701 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2761 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2821 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2881 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2941 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3001 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     3061 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     3121 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     3181 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     3241 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
     3301 gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca ctcatattta
     3361 tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta cgtttgaagt
     3421 acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca ttttttcaca
     3481 ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa agttggtgcg
     3541 catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcgata cgttctctat
     3601 ggaggagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     3661 aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag tcatgtaatt
     3721 agttatgtca cgcttacgtt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg
     3781 agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag
     3841 aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa
     3901 cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcgg
     3961 ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     4021 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4081 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4141 cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4201 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4261 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4321 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4381 gtgatggttc acgtagtggg ccgttgaaca ttcttaggct ggtcgaatca tttagacacg
     4441 ggcatcgtcc tctcgaaagg tggcataggc cactagtgga tctgatatca cctaataact
     4501 tcgtatagca tacattatac gaagttatat taagggttct cgagagctcg ttttcgacac
     4561 tggatggcgg cgttagtatc gaatcgacag cagtatagcg accagcattc acatacgatt
     4621 gacgcatgat attactttct gcgcacttaa cttcgcatct gggcagatga tgtcgaggcg
     4681 aaaaaaaata taaatcacgc taacatttga ttaaaataga acaactacaa tataaaaaaa
     4741 ctatacaaat gacaagttct tgaaaacaag aatcttttta ttgtcagtac tgattagaaa
     4801 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     4861 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     4921 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     4981 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     5041 ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta
     5101 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     5161 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     5221 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     5281 aatacctgga atgctgtttt gccggggatc gcagtggtga gtaaccatgc atcatcagga
     5341 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     5401 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     5461 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     5521 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgaa
     5581 acgtgagtct tttccttacc catggttgtt tatgttcgga tgtgatgtga gaactgtatc
     5641 ctagcaagat tttaaaagga agtatatgaa agaagaacct cagtggcaaa tcctaacctt
     5701 ttatatttct ctacaggggc gcggcgtggg gacaattcaa cgcgtctgtg aggggagcgt
     5761 ttccctgctc gcaggtctgc agcgaggagc cgtaattttt gcttcgcgcc gtgcggccat
     5821 caaaatgtat ggatgcaaat gattatacat ggggatgtat gggctaaatg tacgggcgac
     5881 agtcacatca tgcccctgag ctgcgcacgt caagactgtc aaggagggta ttctgggcct
     5941 ccatgtcgct ggccgggtga cccggcgggg acgaggcaag ctaaacagat ctctagacct
     6001 aataacttcg tatagcatac attatacgaa gttatattaa gggttgtcga cctgcagcgt
     6061 acgaaggtgc ctattgatga tctggcggaa tgtctgccgt gccatagcca tgccttcaca
     6121 tatagt
//
