LOCUS       sequence-346498-        4385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site XII-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4385)
  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 4385)
  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-XII2
FEATURES             Location/Qualifiers
     source          1..4385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      233..821
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     722..741
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1837..1848
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1855..2568
                     /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      2580..2651
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2667..2694
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(3578..4385,1..8))
                     /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(join(3578..4385,1..8))
                     /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     4301..4320
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     join(4385,1..19)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
       61 gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttgt
      121 tgaataaatc gaacttttgc tgagttgaag gatcagtcat gaccaaaatc ccttaacgtg
      181 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      241 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      301 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      361 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
      421 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      481 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      541 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      601 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      661 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      721 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      781 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      841 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
      901 ctgattctgt ggataaccgt gcggccgcca atgcgctgag ggtttaatcg gcatgcaaac
      961 atctacacaa ttagcaaggg caatccatat tttgtctttt cgcgccctgg aaaggcctaa
     1021 gtaatgtcgt aaacgcattc tatctgtact tcaactctcc tctgtgcatt ggtttgtgca
     1081 aatcacattt tacgatactg ccagatatat gcaaaaagag aaaaccaagg gaccagaaca
     1141 aagcaaaatt acgatattct tcgaattcct tcgtgcttga ctaagacaaa gggatggacg
     1201 tagcgatttt tagcgggcca agaactggtt ccgaaaaagc acaggtacac cgaaccctca
     1261 gctaaggagg gacagcaccg atgcggaagg acaaactttc tttttgccta tcacagtatc
     1321 ttatcgagct aactattttc gacacacatg aaaaagcaga aatattaacg aaaaagaaaa
     1381 gaaagaccat gtcatgtacg ggcaatcaga atctgtaaca agcgccattt ttttttctgt
     1441 atcgggccct ccttactgct ctccttccgt gtaacgcgtt atgattaagt cctcagcgag
     1501 ctcgccctga attcgcatct agactgatga gacgtggtag agccacaaac agccggtaca
     1561 agcaacgatc tccaggacca tctgaatcat gcgcggatga cacgaactca cgacggcgat
     1621 cacagacatt aacccacagt acagacactg cgacaacgtg gcaattcgtc gcaatacaac
     1681 gtgagaccga aagtgaaacg tgatttcatg cgtcattttg aacattttgt aaatcttatt
     1741 taataatgtg tgcggcaatt cacatttaat ttatgaatgt tttcttaaca tcgcggcaac
     1801 tcaagaaacg gcaggttcgg atcttagcta ctagagaaag aggagaaata ctagatgcgt
     1861 aaaggcgaag agctgttcac tggtgtcgtc cctattctgg tggaactgga tggtgatgtc
     1921 aacggtcata agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa tggtaaactg
     1981 acgctgaagt tcatctgtac tactggtaaa ctgccggttc cttggccgac tctggtaacg
     2041 acgctgactt atggtgttca gtgctttgct cgttatccgg accatatgaa gcagcatgac
     2101 ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc ctttaaggat
     2161 gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag gcgataccct ggtaaaccgc
     2221 attgagctga aaggcattga ctttaaagag gacggcaata tcctgggcca taagctggaa
     2281 tacaatttta acagccacaa tgtttacatc accgccgata aacaaaaaaa tggcattaaa
     2341 gcgaatttta aaattcgcca caacgtggag gatggcagcg tgcagctggc tgatcactac
     2401 cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc cagacaatca ctatctgagc
     2461 acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg atcatatggt tctgctggag
     2521 ttcgtaaccg cagcgggcat cacgcatggt atggatgaac tgtacaaatg accaggcatc
     2581 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     2641 tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata
     2701 ggtctcagct ggaaatctgc tcgtcagtgg tgctcacact gacgaatcat gtacagatca
     2761 taccgatgac tgcctggcga ctcacaacta agcaagacag ccggaaccag cgccggcgaa
     2821 caccactgca tatatggcat atcacaacag tccacgtctc aagcagttac agagatgtta
     2881 cgaaccacta gtgcactgca gtacaactag agatctatgt gaggatccta actcgagatc
     2941 gagagtcctg caggactagt gctgaggcat taatcgactc tctcgaaatt tttcttaacg
     3001 cgtccttgta ctgcgtctaa cgcttttgcc acttggattt ctattatagg aaatagtctc
     3061 acttactggg cgacgaattt tcgcgttttg atgaagcaca ggaagaattt cttttttttt
     3121 tggcttcttc tggttccgtt ttttacgcgc acaaatctaa aaaaagaaat aattataacc
     3181 tagtctcgaa aattttcatc gatccattcg ttcctttttt tcgatttttt cagatcaaaa
     3241 ttcttgtttc tttctttgtc ttagtttata ttaaaagata ttttgatttt actcctgaac
     3301 tatttattct ttctaagaag gccagaacac tacagctgtt ttaaccgact acgaagttct
     3361 ccattctcga acactagcct tcatttacca aacaggaact agcgtatatc attagtcctt
     3421 attcgaaaag agattggtag atatttattg tagtttgtga gaaggagaaa atactgtcat
     3481 tggactgata gttagaggac attaacctct cttacgttcg ctcattaaga cctcagcgcc
     3541 gagcggccgc gtgttacaac caattaacca attctgatta gaaaaactca tcgagcatca
     3601 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     3661 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     3721 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     3781 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     3841 gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     3901 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgagg cgaaatacgc
     3961 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     4021 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     4081 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     4141 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     4201 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     4261 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     4321 catataaatc agcatccatg ttggaattta atcgcggcct ggagcaagac gtttcccgtt
     4381 gaata
//
