LOCUS       sequence-346538-        4322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site X-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4322)
  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 4322)
  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-X2
FEATURES             Location/Qualifiers
     source          1..4322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      199..787
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     688..707
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1799..1810
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1817..2530
                     /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      2542..2613
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2629..2656
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3481..4296)
                     /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(3481..4296)
                     /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     4204..4223
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4288..4307
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga
       61 gattttgaga cacaacgtgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc
      121 agtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
      181 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
      241 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
      301 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      361 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      421 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      481 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      541 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      601 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      661 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      721 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      781 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      841 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg ccgccaatgc
      901 gctgagggtt taatcgtcta tgaggagact gttagttgga tatcagtaat gagtcgaaaa
      961 agctcgaaat gaatggatat attctttttg ctactggcaa ctgttgaata tttaatgtta
     1021 aaacaaacta actgaggtat attcgtatct gtatgtacac atatactata tacaggaaaa
     1081 gataagcaag agagaggata tcaactacga gagcgatcga ttatatatca aaagctgtcc
     1141 gctttgccac ccataatcgg cgcttagttt cggagttcaa tcataattct accaccttac
     1201 actcaactta ctctttaact cctatagtat aatatcgcca ctgaccccat attaaaaaat
     1261 ttttttgctc gatcttctat cctctttagg ttaattgtcg ctgttattgt ctagattttt
     1321 tctcggagat ggcgcatcta tttgccgtca aaagatcctc tcataccata ttaagtaaat
     1381 tgcctccatt tctttttcct cgggcagaga aactcgcagg caacttgctc tcgaagtggt
     1441 cacgtattaa gtcctcagcg agctcgccct gaattcgcat ctagactgat gagacgtggt
     1501 agagccacaa acagccggta caagcaacga tctccaggac catctgaatc atgcgcggat
     1561 gacacgaact cacgacggcg atcacagaca ttaacccaca gtacagacac tgcgacaacg
     1621 tggcaattcg tcgcaataca acgtgagacc gaaagtgaaa cgtgatttca tgcgtcattt
     1681 tgaacatttt gtaaatctta tttaataatg tgtgcggcaa ttcacattta atttatgaat
     1741 gttttcttaa catcgcggca actcaagaaa cggcaggttc ggatcttagc tactagagaa
     1801 agaggagaaa tactagatgc gtaaaggcga agagctgttc actggtgtcg tccctattct
     1861 ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg
     1921 tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt
     1981 tccttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc
     2041 ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga
     2101 acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga
     2161 aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag aggacggcaa
     2221 tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga
     2281 taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag
     2341 cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct
     2401 gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg
     2461 cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga
     2521 actgtacaaa tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     2581 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg
     2641 ggtgggcctt tctgcgttta taggtctcag ctggaaatct gctcgtcagt ggtgctcaca
     2701 ctgacgaatc atgtacagat cataccgatg actgcctggc gactcacaac taagcaagac
     2761 agccggaacc agcgccggcg aacaccactg catatatggc atatcacaac agtccacgtc
     2821 tcaagcagtt acagagatgt tacgaaccac tagtgcactg cagtacaact agagatctat
     2881 gtgaggatcc taactcgaga tcgagagtcc tgcaggacta gtgctgaggc attaatcctg
     2941 cataatcggc ctcacagagg gatcccgtta cccatctatg ctgaagattt atcatactat
     3001 tcctccgctc gtttcttttt tcagtgaggt gtgtcgtgaa agaaaaccca caattaaact
     3061 ttcaacaacc gggcgactag gaagagagta gtgggcgcgg atgacgaagg ctaaggtcac
     3121 ttcttcgttt cctttattgg ggtttccgtg tagccttccc ctgaatagtg tgggacgttt
     3181 tatgagaagc cgtaagaaat aggcaaattg agttatgaca agtagacatg atgccgcagc
     3241 cttgcctgac tttacgactc cttcatgaat aagtttttct atcgagttct tttccttttt
     3301 tcgccttaat tagctcaatt aagcctgtcc tcactacttt tctttttctt atcggctttg
     3361 tgccacacct aaccttcgaa tgctgtttta ttccgttctt acatgggatg gtaatgcctt
     3421 ggcgagatta agacctcagc gccgagcggc cgcgtgttac aaccaattaa ccaattctga
     3481 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     3541 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     3601 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     3661 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     3721 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     3781 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     3841 cgcctgagcg aggcgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     3901 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3961 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     4021 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     4081 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     4141 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     4201 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     4261 cctggagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     4321 gt
//
