LOCUS       sequence-346515-        4414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site X-3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4414)
  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 4414)
  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: pMC-X3
FEATURES             Location/Qualifiers
     source          1..4414
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      151..739
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     640..659
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1792..1803
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1810..2523
                     /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      2535..2606
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2622..2649
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3525..4340)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3525..4340)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4248..4267
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4332..4351
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tgtaacatca gagattttga gacacaacgt ggctttgttg aataaatcga acttttgctg
       61 agttgaagga tcagtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
      121 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
      181 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
      241 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
      301 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
      361 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
      421 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
      481 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      541 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      601 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      661 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
      721 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
      781 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtgc
      841 ggccgccaat gcgctgaggg tttaatcgag atctttgtgt tcggttaccc ggctcagatc
      901 ctaacttctt cttttggtat gtttattcgt ataagttact gttgtccaca ggcaatactc
      961 tgcagaaaat taaaacggca ttaatgctag gacaaccaga attgttacta ctgtatgtgc
     1021 gatagttgat aactgcaaca ttatgcccgg tatattctca aaaaacccta ttactgcata
     1081 cgaagaaatc gcaagagaaa tctttcggtt tggaaaagct cactgtgagg ttccttggag
     1141 ccaatagtaa tacagcacaa tccaaggaaa aatctggcct atatgcaagg aaggagagat
     1201 agtcaaaagc attctttccc ctagaagttg gtgcatatat ggcatcgtta aaacatatta
     1261 cccccaaaat ttcttctcta aacgatgtgc ttggcctttg ttttggtttt tgatgtcggt
     1321 cgtttgaggc cccttgcgga aaatcgagat cgccgaatgg cacgcgaggg aagggaaata
     1381 aggtttaaag gcactgaaac aataggcaag aagtaggcga gagccgacat acgagactat
     1441 taagtcctca gcgagctcgc cctgaattcg catctagact gatgagacgt ggtagagcca
     1501 caaacagccg gtacaagcaa cgatctccag gaccatctga atcatgcgcg gatgacacga
     1561 actcacgacg gcgatcacag acattaaccc acagtacaga cactgcgaca acgtggcaat
     1621 tcgtcgcaat acaacgtgag accgaaagtg aaacgtgatt tcatgcgtca ttttgaacat
     1681 tttgtaaatc ttatttaata atgtgtgcgg caattcacat ttaatttatg aatgttttct
     1741 taacatcgcg gcaactcaag aaacggcagg ttcggatctt agctactaga gaaagaggag
     1801 aaatactaga tgcgtaaagg cgaagagctg ttcactggtg tcgtccctat tctggtggaa
     1861 ctggatggtg atgtcaacgg tcataagttt tccgtgcgtg gcgagggtga aggtgacgca
     1921 actaatggta aactgacgct gaagttcatc tgtactactg gtaaactgcc ggttccttgg
     1981 ccgactctgg taacgacgct gacttatggt gttcagtgct ttgctcgtta tccggaccat
     2041 atgaagcagc atgacttctt caagtccgcc atgccggaag gctatgtgca ggaacgcacg
     2101 atttccttta aggatgacgg cacgtacaaa acgcgtgcgg aagtgaaatt tgaaggcgat
     2161 accctggtaa accgcattga gctgaaaggc attgacttta aagaggacgg caatatcctg
     2221 ggccataagc tggaatacaa ttttaacagc cacaatgttt acatcaccgc cgataaacaa
     2281 aaaaatggca ttaaagcgaa ttttaaaatt cgccacaacg tggaggatgg cagcgtgcag
     2341 ctggctgatc actaccagca aaacactcca atcggtgatg gtcctgttct gctgccagac
     2401 aatcactatc tgagcacgca aagcgttctg tctaaagatc cgaacgagaa acgcgatcat
     2461 atggttctgc tggagttcgt aaccgcagcg ggcatcacgc atggtatgga tgaactgtac
     2521 aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     2581 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     2641 ctttctgcgt ttataggtct cagctggaaa tctgctcgtc agtggtgctc acactgacga
     2701 atcatgtaca gatcataccg atgactgcct ggcgactcac aactaagcaa gacagccgga
     2761 accagcgccg gcgaacacca ctgcatatat ggcatatcac aacagtccac gtctcaagca
     2821 gttacagaga tgttacgaac cactagtgca ctgcagtaca actagagatc tatgtgagga
     2881 tcctaactcg agatcgagag tcctgcagga ctagtgctga ggcattaatc cacttttcaa
     2941 tgaaacggat attgatatgc tagtaaaagg acgagctcaa gagcgaaaat ataagtaaag
     3001 aattcgagtg cacttgtctc catgcagcaa gatttcatat gagtcttttt tatcttttta
     3061 ctttttacat tacacgatat gcactttatg aaaatttaac gaggttggaa gccggataat
     3121 caaccaaaat caggcacgaa ggcacactcg tatatgcatg ttgttgaaac tctgttacgc
     3181 tgaactaaca atcacacatg tagaggtcac cgggaaaagt tgcgacccca tggaaggtcg
     3241 atctcttcgt ttggctttgc ttggctggcg gcattgcgct tcttcgctta tacccgactc
     3301 ttgacgctcg agctcgttca ttgagatacc tttattcttg cacattttct ggcttttttc
     3361 gctactcggg tacatgtaat catgcacaca gaaggtgctg tagggtgaaa gttcctttgt
     3421 gctgtcgttt gtttttaatg ccaaactttc cggtgatcaa taaccacctc attaagacct
     3481 cagcgccgag cggccgcgtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg
     3541 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     3601 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     3661 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     3721 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     3781 ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     3841 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgaggcga
     3901 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     3961 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     4021 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     4081 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     4141 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     4201 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     4261 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctgga gcaagacgtt
     4321 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     4381 attgttcatg atgatatatt tttatcttgt gcaa
//
