LOCUS       sequence-265270-        6352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hph 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 6352)
  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 6352)
  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: pMEL12
FEATURES             Location/Qualifiers
     source          1..6352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(556..561,562..579,580..586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    594..610
                     /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     599..621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     618..634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     618..634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     653..673
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        655..673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        691..959
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        980..1054
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1059..1078
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      1084..1331
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1349..1368)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1350..1368)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1378..1395)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1378..1394)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1387..1409)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1622..1641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(1868..1901)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(1919..2166)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     CDS             complement(2174..3202)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(2174..3202)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(3203..3546)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    complement(3601..3634)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3823..3870
                     /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      3851..4833
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        4860..4964
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4965..5033,5034..5825)
                     /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(5183..5202)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5996..6352,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
      661 ccctcactaa agggaacaaa agctggagct tctttgaaaa gataatgtat gattatgctt
      721 tcactcatat ttatacagaa acttgatgtt ttctttcgag tatatacaag gtgattacat
      781 gtacgtttga agtacaactc tagattttgt agtgccctct tgggctagcg gtaaaggtgc
      841 gcattttttc acaccctaca atgttctgtt caaaagattt tggtcaaacg ctgtagaagt
      901 gaaagttggt gcgcatgttt cggcgttcga aacttctccg cagtgaaaga taaatgatcg
      961 atacgttctc tatggaggag ttttagagct agaaatagca agttaaaata aggctagtcc
     1021 gttatcaact tgaaaaagtg gcaccgagtc ggtggtgctt tttttgtttt ttatgtcttc
     1081 gagtcatgta attagttatg tcacgcttac gttcacgccc tccccccaca tccgctctaa
     1141 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     1201 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     1261 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     1321 ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     1381 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     1441 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     1501 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
     1561 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1621 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1681 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1741 aaacttgatt agggtgatgg ttcacgtagt gggccgttga acattcttag gctggtcgaa
     1801 tcatttagac acgggcatcg tcctctcgaa aggtgacata ggccactagt ggatctgata
     1861 tcacctaata acttcgtata gcatacatta tacgaagtta tattaagggt tctcgagagc
     1921 tcgttaaagc cttcgagcgt cccaaaacct tctcaagcaa ggttttcagt ataatgttac
     1981 atgcgtacac gcgtctgtac agaaaaaaaa gaaaaatttg aaatataaat aacgttctta
     2041 atactaacat aactataaaa aaataaatag ggacctagac ttcaggttgt ctaactcctt
     2101 ccttttcggt tagagcggat gtggggggag ggcgtgaatg taagcgtgac ataactaatt
     2161 acatgactcg agattattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat
     2221 cggcgagtac ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg
     2281 tacgcccgac agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag
     2341 ctgcatcatc gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga
     2401 gcatatacgc ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc
     2461 gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt
     2521 attgggaatc cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt
     2581 ccgtcaggac attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt
     2641 cctcggccca aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt
     2701 ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc
     2761 atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa
     2821 gatcggccgc agcgatcgca tccatggcct ccgcgaccgg ctgcagaaca gcgggcagtt
     2881 cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca
     2941 ggctctcgct gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa
     3001 agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga
     3061 catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct
     3121 gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg
     3181 tgagttcagg ctttttaccc atggttgttt atgttcggat gtgatgtgag aactgtatcc
     3241 tagcaagatt ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat cctaaccttt
     3301 tatatttctc tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt
     3361 tccctgctcg caggtccgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc
     3421 aaaatgtatg gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca
     3481 gtcacatcat gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc
     3541 catgtcgctg gccgggtgac ccggcgggga cgaggcaagc taaacagatc tctagaccta
     3601 ataacttcgt atagcataca ttatacgaag ttatattaag ggttgtcgac ctgcagcgta
     3661 cgaaggtgcc tattgatgat ctggcggaat gtctgccgtg ccatagccat gccttcacat
     3721 atagtacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc
     3781 tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc tattccgaag
     3841 ttcctattct ctagaaagta taggaacttc agagcgcttt tgaaaaccaa aagcgctctg
     3901 aagacgcact ttcaaaaaac caaaaacgca ccggactgta acgagctact aaaatattgc
     3961 gaataccgct tccacaaaca ttgctcaaaa gtatctcttt gctatatatc tctgtgctat
     4021 atccctatat aacctaccca tccacctttc gctccttgaa cttgcatcta aactcgacct
     4081 ctacattttt tatgtttatc tctagtatta ctctttagac aaaaaaattg tagtaagaac
     4141 tattcataga gtgaatcgaa aacaatacga aaatgtaaac atttcctata cgtagtatat
     4201 agagacaaaa tagaagaaac cgttcataat tttctgacca atgaagaatc atcaacgcta
     4261 tcactttctg ttcacaaagt atgcgcaatc cacatcggta tagaatataa tcggggatgc
     4321 ctttatcttg aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc gggaagtgga
     4381 gtcaggcttt ttttatggaa gagaaaatag acaccaaagt agccttcttc taaccttaac
     4441 ggacctacag tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa aggagaaaaa
     4501 aagtaatcta agatgctttg ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca
     4561 aaaaagaagt atagattctt tgttggtaaa atagcgctct cgcgttgcat ttctgttctg
     4621 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
     4681 ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg
     4741 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt
     4801 ttgttctaca aaatgaagca cagatgcttc gttcaggtgg cacttttcgg ggaaatgtgc
     4861 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4921 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4981 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5041 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5101 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5161 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5221 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5281 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5341 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5401 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5461 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5521 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5581 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5641 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5701 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5761 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5821 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5881 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5941 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6001 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6061 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6121 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6181 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6241 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6301 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga ac
//
