LOCUS       sequence-346542-        4412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site XI-3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4412)
  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 4412)
  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-XI3
FEATURES             Location/Qualifiers
     source          1..4412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      225..813
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     714..733
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1869..1880
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1887..2600
                     /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      2612..2683
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2699..2726
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3597..4412)
                     /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(3597..4412)
                     /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     4320..4339
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     join(4404..4412,1..11)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
       61 tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt gttgaataaa
      121 tcgaactttt gctgagttga aggatcagtc atgaccaaaa tcccttaacg tgagttttcg
      181 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
      241 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
      301 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
      361 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
      421 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
      481 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
      541 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
      601 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
      661 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
      721 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
      781 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
      841 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
      901 gtggataacc gtgcggccgc caatgcgctg agggtttaat agttacttgc tctatgcgtt
      961 tgcgcatcct ctttttactt tttttttttc agtaaagcct aagcataaat cgttttatac
     1021 gtacgacacg ttcaactttt cttggttagt agtggcaatc tctgcaatac atacagggag
     1081 tcatggtcta tcatcttgtc caatcaaaga agcatcggtt cagatcgagc aaactgtagg
     1141 gagaaaggaa agtagaaatg cagagtgtgc tatatgtcca atctcggttt tgtagtttgg
     1201 atgtcattag agatctacca cccaaccggc tgctttcatg tggaacagaa aagaaatcgg
     1261 ggcgcttcct cttctgtatt cctttagtta acgtttttat tcagccatct aaccatcata
     1321 cccccatacg gtaacaaaac ctcttctaag aaaagaagtc tctgctcctc cgccatctta
     1381 tttttattcg ctgcgcgcgt ttattgtcgc atcgctggcc agcaaaaaat tggttgcctt
     1441 tttttaccta aaaaagacac atctaactga ttagttttcc gttttaggat attgacgcca
     1501 agcgtgcgtc tgattattaa gtcctcagcg agctcgccct gaattcgcat ctagactgat
     1561 gagacgtggt agagccacaa acagccggta caagcaacga tctccaggac catctgaatc
     1621 atgcgcggat gacacgaact cacgacggcg atcacagaca ttaacccaca gtacagacac
     1681 tgcgacaacg tggcaattcg tcgcaataca acgtgagacc gaaagtgaaa cgtgatttca
     1741 tgcgtcattt tgaacatttt gtaaatctta tttaataatg tgtgcggcaa ttcacattta
     1801 atttatgaat gttttcttaa catcgcggca actcaagaaa cggcaggttc ggatcttagc
     1861 tactagagaa agaggagaaa tactagatgc gtaaaggcga agagctgttc actggtgtcg
     1921 tccctattct ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg
     1981 agggtgaagg tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta
     2041 aactgccggt tccttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg
     2101 ctcgttatcc ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct
     2161 atgtgcagga acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag
     2221 tgaaatttga aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag
     2281 aggacggcaa tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca
     2341 tcaccgccga taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg
     2401 aggatggcag cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc
     2461 ctgttctgct gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga
     2521 acgagaaacg cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg
     2581 gtatggatga actgtacaaa tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     2641 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg
     2701 ctcaccttcg ggtgggcctt tctgcgttta taggtctcag ctggaaatct gctcgtcagt
     2761 ggtgctcaca ctgacgaatc atgtacagat cataccgatg actgcctggc gactcacaac
     2821 taagcaagac agccggaacc agcgccggcg aacaccactg catatatggc atatcacaac
     2881 agtccacgtc tcaagcagtt acagagatgt tacgaaccac tagtgcactg cagtacaact
     2941 agagatctat gtgaggatcc taactcgaga tcgagagtcc tgcaggacta gtgctgaggc
     3001 attaatttac gtggattgag ccagcaatac agatcattat taaattgttt tgtacatgat
     3061 gttagtatat aatcgtaaag cttttctaat atgtatacct tatacatgga actccacaga
     3121 acttgcaaac ataccaaaaa tcccttattc ttgttcactc attttacatc aaaaaataat
     3181 atttcagtta ttaaggaaaa taaaaaaata gattagagaa gcattttgaa gaaatagtat
     3241 attcttttat tgaacctaag agcgtgatat ttttactcga aataaaatac gaaaaatcta
     3301 tgcactcatc tttccgacta ctattggctc ctgctcaaaa aaagagggaa aaaaagctcc
     3361 aaaattctat cttttcctat cgctcctgtc ctatccttat tacgttcatt actattttaa
     3421 tactatccat tcttttattt tcagtctaaa aaaaacattt ctcataacgg gaaaagcaaa
     3481 aaaatgtcaa gcttatacat caaaacacca ctgcatgcat tatctgctgg tccggattct
     3541 caattaagac ctcagcgccg agcggccgcg tgttacaacc aattaaccaa ttctgattag
     3601 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     3661 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     3721 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     3781 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     3841 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     3901 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     3961 tgagcgaggc gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     4021 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     4081 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     4141 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     4201 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     4261 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     4321 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctg
     4381 gagcaagacg tttcccgttg aatatggctc at
//
