LOCUS       sequence-346553-        5589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2 mu cloning vector for single or multiplexed gRNAs, 
            SNR52p-sfGFP-scRNA-SUP4t-CYC1t.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5589)
  AUTHORS   Otto M, Skrekas C, Gossing M, Gustafsson J, Siewers V, David F
  TITLE     Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based 
            Applications, Genomic Integrations and Combinatorial Libraries.
  JOURNAL   ACS Synth Biol. 2021 Dec 17;10(12):3461-3474. doi: 
            10.1021/acssynbio.1c00408. Epub 2021 Dec 3.
  PUBMED    34860007
REFERENCE   2  (bases 1 to 5589)
  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"; doi: 
            "10.1021/acssynbio.1c00408"; journalName: "ACS Synth Biol"; date: 
            "2021-12-17- 17"; volume: "10"; issue: "12"; pages: "3461-3474"
COMMENT     Sequence Label: pMCL9
FEATURES             Location/Qualifiers
     source          1..5589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(77..181)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      284..872
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     773..792
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        984..1252
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     RBS             1408..1419
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1426..2139
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2151..2222
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2238..2265
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        2279..2353
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2358..2377
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      2383..2630
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     CDS             2812..3615
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      3698..4680
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(join(4805..5589,1..7,8..76))
                     /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     5428..5447
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
       61 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
      121 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
      181 gcacatttcc ccgaaaagtg ccacctgtca tgaccaaaat cccttaacgt gagttttcgt
      241 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
      301 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
      361 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
      421 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
      481 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
      541 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
      601 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
      661 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
      721 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
      781 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
      841 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
      901 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
      961 tggataaccg tgcggccgcc ccttctttga aaagataatg tatgattatg ctttcactca
     1021 tatttataca gaaacttgat gttttctttc gagtatatac aaggtgatta catgtacgtt
     1081 tgaagtacaa ctctagattt tgtagtgccc tcttgggcta gcggtaaagg tgcgcatttt
     1141 ttcacaccct acaatgttct gttcaaaaga ttttggtcaa acgctgtaga agtgaaagtt
     1201 ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa agataaatga tctgagacgg
     1261 aaagtgaaac gtgatttcat gcgtcatttt gaacattttg taaatcttat ttaataatgt
     1321 gtgcggcaat tcacatttaa tttatgaatg ttttcttaac atcgcggcaa ctcaagaaac
     1381 ggcaggttcg gatcttagct actagagaaa gaggagaaat actagatgcg taaaggcgaa
     1441 gagctgttca ctggtgtcgt ccctattctg gtggaactgg atggtgatgt caacggtcat
     1501 aagttttccg tgcgtggcga gggtgaaggt gacgcaacta atggtaaact gacgctgaag
     1561 ttcatctgta ctactggtaa actgccggtt ccttggccga ctctggtaac gacgctgact
     1621 tatggtgttc agtgctttgc tcgttatccg gaccatatga agcagcatga cttcttcaag
     1681 tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt cctttaagga tgacggcacg
     1741 tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg cattgagctg
     1801 aaaggcattg actttaaaga ggacggcaat atcctgggcc ataagctgga atacaatttt
     1861 aacagccaca atgtttacat caccgccgat aaacaaaaaa atggcattaa agcgaatttt
     1921 aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac
     1981 actccaatcg gtgatggtcc tgttctgctg ccagacaatc actatctgag cacgcaaagc
     2041 gttctgtcta aagatccgaa cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc
     2101 gcagcgggca tcacgcatgg tatggatgaa ctgtacaaat gaccaggcat caaataaaac
     2161 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     2221 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat acgtctcagt
     2281 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     2341 caccgagtcg gtggtgcttt ttttgttttt tatgtcttcg agtcatgtaa ttagttatgt
     2401 cacgcttacg ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac
     2461 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     2521 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
     2581 tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc tacacggttt
     2641 ccttgaaatt tttttgattc ggtaatctcc gaacagaagg aagaacgaag gaaggagcac
     2701 agacttagat tggtatatat acgcatatgt agtgttgaag aaacatgaaa ttgcccagta
     2761 ttcttaaccc aactgcacag aacaaaaacg tgcaggaaac gaagataaat catgtcgaaa
     2821 gctacatata aggaacgtgc tgctactcat cctagtcctg ttgctgccaa gctatttaat
     2881 atcatgcacg aaaagcaaac aaacttgtgt gcttcattgg atgttcgtac caccaaggaa
     2941 ttactggagt tagttgaagc attaggtccc aaaatttgtt tactaaaaac acatgtggat
     3001 atcttgactg atttttccat ggagggcaca gttaagccgc taaaggcatt atccgccaag
     3061 tacaattttt tactcttcga ggacagaaaa tttgctgaca ttggtaatac agtcaaattg
     3121 cagtactctg cgggtgtata cagaatagca gaatgggcag acattacgaa tgcacacggt
     3181 gtggtgggcc caggtattgt tagcggtttg aagcaggcgg cagaagaagt aacaaaggaa
     3241 cctagaggcc ttttgatgtt agcagaattg tcatgcaagg gctccctatc tactggagaa
     3301 tatactaagg gtactgttga cattgcgaag agcgacaaag attttgttat cggctttatt
     3361 gctcaaagag acatgggtgg aagagatgaa ggttacgatt ggttgattat gacacccggt
     3421 gtgggtttag atgacaaggg agatgcattg ggtcaacagt atagaaccgt ggatgatgtg
     3481 gtttctacag gatctgacat tattattgtt ggaagaggac tatttgcaaa gggaagggat
     3541 gctaaggtag agggtgaacg ttacagaaaa gcaggctggg aagcatattt gagaagatgc
     3601 ggccagcaaa actaaaaaac tgtattataa gtaaatgcat gtatactaaa ctcacaaatt
     3661 agagcttcaa tttaattata tcagttatta cccgagtaac gaagcatctg tgcttcattt
     3721 tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat
     3781 ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc
     3841 atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc
     3901 tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat
     3961 acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct
     4021 tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca
     4081 ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa
     4141 aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt
     4201 caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa
     4261 cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta
     4321 ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac
     4381 tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata
     4441 aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt
     4501 atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga
     4561 agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa
     4621 agtgcgtcat cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag
     4681 ctagagaata ggaacttccc gagcggccgc gattatcaaa aaggatcttc acctagatcc
     4741 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4801 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4861 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4921 gccccagtgc tgcaatgata ccgcgggacc cacgctcacc ggctccagat ttatcagcaa
     4981 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5041 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5101 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5161 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5221 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5281 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5341 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5401 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5461 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5521 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5581 ccagcgttt
//
