LOCUS       sequence-346535-        6612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-1 backbone with toxic gene dropout 
            cassette (ccdB and TPK2) and URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6612)
  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 6612)
  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: pMG343
FEATURES             Location/Qualifiers
     source          1..6612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             351..1154
                     /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"
     misc_feature    1237..1740
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(join(1851..2642,2643..2711))
                     /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     2474..2493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2712..2816)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2919..3507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3408..3427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3821..4228
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             5978..6283
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     6162..6181
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
ORIGIN
        1 ccaatgagac gacggggtca tcacggctca tcatgcgcca aacaaatgtg tgcaatacac
       61 gctcggatga ctgcatgatg accgcactga ctggggacag cagatccacc taagcctgtg
      121 agagaagcag acacccgaca gatcaaggca gttaactagt gcactgcagt acacggtttc
      181 cttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
      241 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
      301 tcttaaccca actgcacaga acaaaaacgt gcaggaaacg aagataaatc atgtcgaaag
      361 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
      421 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
      481 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
      541 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
      601 acaatttttt actcttcgag gacagaaaat ttgctgacat tggtaataca gtcaaattgc
      661 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg
      721 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac
      781 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat
      841 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg
      901 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg
      961 tgggtttaga tgacaaggga gatgcattgg gtcaacagta tagaaccgtg gatgatgtgg
     1021 tttctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg
     1081 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg
     1141 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta
     1201 gagcttcaat ttaattatat cagttattac ccgagtatca cgtgctataa aaataattat
     1261 aatttaaatt ttttaatata aatatataaa ttaaaaatag aaagtaaaaa aagaaattaa
     1321 agaaaaaata gtttttgttt tccgaagatg taaaagactc tagggggatc gccaacaaat
     1381 actacctttt atcttgctct tcctgctctc aggtattaat gccgaattgt ttcatcttgt
     1441 ctgtgtagaa aaccacacac gaaaatcctg tgattttaca ttttacttat cgttaatcga
     1501 atgtatatct atttaatctg cttttcttgt ctaataaata tatatgtaaa gtacgctttt
     1561 tgttgaaatt ttttaaacct ttgtttattt ttttttcttc attccgtaac tcttctacct
     1621 tctttattta ctttctaaaa tccaaataca aaacataaaa ataaataaac acagagtaaa
     1681 ttcccaaatt attccatcat taaaagatac gaggcgcgtg taagttacag gcaagcgatc
     1741 cgtcccgagc ggccgcgatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1801 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     1861 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     1921 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     1981 atgataccgc gggacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2041 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2101 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2161 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     2221 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2281 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2341 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     2401 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     2461 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     2521 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2581 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2641 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2701 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     2761 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     2821 tttccccgaa aagtgccacc tgtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2881 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2941 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3001 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3061 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3121 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3181 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3241 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3301 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3361 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3421 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3481 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3541 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3601 aaccgtgcgg ccgcccctga attcgcatct agatggtaga gccacaaaca gccggtacaa
     3661 gcaacgatct ccaggaccat ctgaatcatg cgcggatgac acgaactcac gacggcgatc
     3721 acagacatta acccacagta cagacactgc gacaacgtgg caattcgtcg caataccgtc
     3781 tcactgaact ggccgataat tgcagacgaa cgtgagaccc ccacacacca tagcttcaaa
     3841 atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca
     3901 cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt
     3961 taattacccg tactaaaggt ttggaaaaga aaaaagacac cgcctcgttt ctttttcttc
     4021 gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaaatt tttttttttg
     4081 atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg tcatcaattt
     4141 ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc ttgctcatta
     4201 gaaagaaagc atagcaatct aatctaagtt ttaattacaa aagatctatg gaattcgttg
     4261 cagaaagggc tcagccagtt ggtcaaacaa tccagcagca aaatgttaat acttacgggc
     4321 aaggcgtcct acaaccgcat catgatttac agcagcgaca acaacaacaa cagcagcgtc
     4381 agcatcaaca actgctgacg tcacagttgc cccagaaatc tctcgtatcc aaaggcaaat
     4441 atacactaca tgacttccag attatgagaa cgcttggtac tggatctttt ggtagggttc
     4501 atttggtgcg ctctgttcac aatggtcggt attatgctat aaaagtttta aagaaacaac
     4561 aagttgtcaa gatgaaacag gttgaacata ccaatgacga acgacgtatg ctaaagcttg
     4621 tggagcatcc gtttctgatt agaatgtggg gtacgtttca agatgctagg aatatcttta
     4681 tggtgatgga ttatatcgaa ggtggtgaac ttttctcgtt actgagaaag tcacaaagat
     4741 ttcctaatcc tgtagcaaaa ttttacgctg cggaagtcat actggcatta gagtacttgc
     4801 atgctcataa tatcatctac agggatttaa agccagaaaa tatcttgctg gatagaaatg
     4861 gccacattaa aataaccgat tttgggttcg ccaaagaggt acaaactgtc acatggacgc
     4921 tttgtgggac tcctgattac attgctcctg aagttattac cacaaagcca tataataagt
     4981 cggtggattg gtggtcccta ggtgttctaa tctacgaaat gttagctggt tatacaccct
     5041 tttacgatac tactccaatg aagacgtatg aaaagatttt acaaggtaaa gtagtatatc
     5101 caccatattt tcatcccgac gtcgtggatc tactaagcaa actgatcact gcagatttga
     5161 caagaagaat cggtaattta caaagcggtt ccagggacat caaagctcac ccatggttta
     5221 gtgaagttgt atgggaaaga ttgttagcaa aggatattga aactccatac gagcctccta
     5281 tcacatcagg tatcggtgac acgtctttat tcgatcaata tcccgaggag cagctagatt
     5341 atggtattca aggcgatgat ccatatgctg aatactttca agatttctaa ggatcctaac
     5401 tcgagataaa gcaatcttga tgaggataat gatttttttt tgaatataca taaatactac
     5461 cgtttttctg ctagattttg tgatgacgta aataagtaca tattactttt taagccaaga
     5521 caagattaag cattaacttt acccttttct ttctaagttt caatattagt tatcactgtt
     5581 taaaagttat ggcgagaacg tcggcggtta aaatatatta ccctgaacgg ctgacgccgc
     5641 gtggatccgg cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg
     5701 gtataagaat atatactgat atgtataccc gaagtatgtc aaaaagaggt atgctatgaa
     5761 gcagcgtatt acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat
     5821 gtcaatatct ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc
     5881 gaacgctgga aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg
     5941 aacggctctt ttgctgacga gaacaggggc tggtgaaatg cagtttaagg tttacaccta
     6001 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc
     6061 cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg
     6121 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat
     6181 ggccagtgtg ccggtttccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     6241 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat
     6301 acacagccag tctgcaggtc gatacagtag aaattacaga aactttatca cgtttagtaa
     6361 gtatagaggc tgaaaatcca gatgaagccg aacgacttgt aagagaaaag tataagagtt
     6421 gtgaaattgt tcttgatgca gatgattttc aggactatga cactagcgta tatgaatagg
     6481 tagatgtttt tattttgtca cacaaaaaag aggctcgcac ctctttttct tatttctttt
     6541 tatgatttaa tacggcattg aggacaatag cgagtaggct gcgatcgcac gcattccgtt
     6601 gggtctcagc tg
//
