LOCUS       sequence-353632-        4431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site XII-5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4431)
  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 4431)
  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-XII5
FEATURES             Location/Qualifiers
     source          1..4431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      230..818
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     719..738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1753..1764
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1771..2484
                     /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      2496..2567
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2583..2610
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(3621..4431,1..5))
                     /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(3621..4431,1..5))
                     /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     4344..4363
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     join(4428..4431,1..16)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
       61 atatttttat cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga
      121 ataaatcgaa cttttgctga gttgaaggat cagtcatgac caaaatccct taacgtgagt
      181 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
      241 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
      301 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
      361 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
      421 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
      481 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
      541 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
      601 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
      661 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
      721 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
      781 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
      841 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
      901 attctgtgga taaccgtgcg gccgccaatg cgctgagggt ttaatcaatc tggcggcttg
      961 agttctcaac atgttttatt ttttacttat attgctggta gggtaaaaaa atataactcc
     1021 taggaatagg ttgtctatat gtttttgtct tgcttctata attgtaacaa acaaggaaag
     1081 ggaaaatact gggtgtaaaa gccattgagt caagttaggt catccctttt atacaaaatt
     1141 tttcaatttt ttttccaaga ttcttgtacg attaattatt ttttttttgc gtcctacagc
     1201 gtgatgaaaa tttcgcctgc tgcaagatga gcgggaacgg gcgaaatgtg cacgcgcaca
     1261 acttacgaaa cgcggatgag tcactgacag ccaccgcaga ggttctgact cctactgagc
     1321 tctattggag gtggcagaac cggtaccgga ggaggccgct ataaccggtt tgaatttatt
     1381 gtcacagtgt cacatcagca ttaagtcctc agcgagctcg ccctgaattc gcatctagac
     1441 tgatgagacg tggtagagcc acaaacagcc ggtacaagca acgatctcca ggaccatctg
     1501 aatcatgcgc ggatgacacg aactcacgac ggcgatcaca gacattaacc cacagtacag
     1561 acactgcgac aacgtggcaa ttcgtcgcaa tacaacgtga gaccgaaagt gaaacgtgat
     1621 ttcatgcgtc attttgaaca ttttgtaaat cttatttaat aatgtgtgcg gcaattcaca
     1681 tttaatttat gaatgttttc ttaacatcgc ggcaactcaa gaaacggcag gttcggatct
     1741 tagctactag agaaagagga gaaatactag atgcgtaaag gcgaagagct gttcactggt
     1801 gtcgtcccta ttctggtgga actggatggt gatgtcaacg gtcataagtt ttccgtgcgt
     1861 ggcgagggtg aaggtgacgc aactaatggt aaactgacgc tgaagttcat ctgtactact
     1921 ggtaaactgc cggttccttg gccgactctg gtaacgacgc tgacttatgg tgttcagtgc
     1981 tttgctcgtt atccggacca tatgaagcag catgacttct tcaagtccgc catgccggaa
     2041 ggctatgtgc aggaacgcac gatttccttt aaggatgacg gcacgtacaa aacgcgtgcg
     2101 gaagtgaaat ttgaaggcga taccctggta aaccgcattg agctgaaagg cattgacttt
     2161 aaagaggacg gcaatatcct gggccataag ctggaataca attttaacag ccacaatgtt
     2221 tacatcaccg ccgataaaca aaaaaatggc attaaagcga attttaaaat tcgccacaac
     2281 gtggaggatg gcagcgtgca gctggctgat cactaccagc aaaacactcc aatcggtgat
     2341 ggtcctgttc tgctgccaga caatcactat ctgagcacgc aaagcgttct gtctaaagat
     2401 ccgaacgaga aacgcgatca tatggttctg ctggagttcg taaccgcagc gggcatcacg
     2461 catggtatgg atgaactgta caaatgacca ggcatcaaat aaaacgaaag gctcagtcga
     2521 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca
     2581 ctggctcacc ttcgggtggg cctttctgcg tttataggtc tcagctggaa atctgctcgt
     2641 cagtggtgct cacactgacg aatcatgtac agatcatacc gatgactgcc tggcgactca
     2701 caactaagca agacagccgg aaccagcgcc ggcgaacacc actgcatata tggcatatca
     2761 caacagtcca cgtctcaagc agttacagag atgttacgaa ccactagtgc actgcagtac
     2821 aactagagat ctatgtgagg atcctaactc gagatcgaga gtcctgcagg actagtgctg
     2881 aggcattaat gcaactcaga agtttgacag caagcaagtt catcattcga actagcctta
     2941 ttgttttagt tcagtgacag cgaactgccg tactcgatgc tttatttctc acggtagagc
     3001 ggaagaacag ataggggcag cgtgagaaga gttagaaagt aaatttttat cacgtctgaa
     3061 gtattcttat tcataggaaa ttttgcaagg ttttttagct caataacggg ctaagttata
     3121 taaggtgttc acgcgatttt cttgttatgt atacctcttc tctgaggaat ggtactactg
     3181 tcctgatgta ggctccttaa attggtgggc aagaataact tatcgatatt ttgtatattg
     3241 gtcttggagt tcaccacgta atgcctgttt aagaccatca gttaactcta gtattatttg
     3301 gtcttggcta ctggccgttt gctattattc aagtcttttg tgccttcccg tcgggtaagg
     3361 gagttattta gggatacaga atctaacgaa aactaaatct caatgattaa ctctatttaa
     3421 tccttttttg aaaggcaaaa gaggtccctt gttcacttac aacgttctta gccaaattcg
     3481 cttatcactt actacttcac gatatacaga agtaaaaaca tataaaaaga tgtctgtttg
     3541 tttagccatc acaaaaggta tcgcagatta agacctcagc gccgagcggc cgcgtgttac
     3601 aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     3661 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     3721 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     3781 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     3841 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc
     3901 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     3961 ccgttattca ttcgtgattg cgcctgagcg aggcgaaata cgcgatcgct gttaaaagga
     4021 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     4081 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     4141 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     4201 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     4261 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     4321 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     4381 atgttggaat ttaatcgcgg cctggagcaa gacgtttccc gttgaatatg g
//
