LOCUS       sequence-346536-        4269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site X-4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4269)
  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 4269)
  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-X4
FEATURES             Location/Qualifiers
     source          1..4269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     211..230
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1313..1324
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1331..2044
                     /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      2056..2127
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2143..2170
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2951..3766)
                     /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(2951..3766)
                     /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     3674..3693
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3758..3777
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(3991..4269,1..310)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
       61 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
      121 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      181 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      241 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      301 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      361 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgtg cggccgccaa
      421 tgcgctgagg gtttaatccc aaagctaaga gtcccatttt attcttctat atgtatattt
      481 tcgatactct aaaccaccct acaatgtagc cctatactaa atctgctcaa ttttcagctt
      541 ctacaagtga ctcgaggcca cgtggaaaga tccaactact ccagcacaac gattcaatat
      601 aatcgattgc tccactcata agaggcaaga acaagcttca acttttggta agccgccgtt
      661 tataaacagg gaagatgtcc tttgtcaagg gaggcacaga gcatggccaa tttggcaaat
      721 tgcaggtttt tctgagtgaa aaatgaaaaa gcattgtagt agagtcggct cactgaaaaa
      781 ccggggagga cgaaaaggtt tccagccaca gttgtagtca cgtgcgcgcc atgctgacta
      841 atggcagccg tcgttgggca gaagagaatt agtatggtac aggatacgct aattgcgctc
      901 caactaccaa ggttgttgag ggaacactgg ggcaataggc tgtcgccatt caagagcaga
      961 ttaagtcctc agcgagctcg ccctgaattc gcatctagac tgatgagacg tggtagagcc
     1021 acaaacagcc ggtacaagca acgatctcca ggaccatctg aatcatgcgc ggatgacacg
     1081 aactcacgac ggcgatcaca gacattaacc cacagtacag acactgcgac aacgtggcaa
     1141 ttcgtcgcaa tacaacgtga gaccgaaagt gaaacgtgat ttcatgcgtc attttgaaca
     1201 ttttgtaaat cttatttaat aatgtgtgcg gcaattcaca tttaatttat gaatgttttc
     1261 ttaacatcgc ggcaactcaa gaaacggcag gttcggatct tagctactag agaaagagga
     1321 gaaatactag atgcgtaaag gcgaagagct gttcactggt gtcgtcccta ttctggtgga
     1381 actggatggt gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc
     1441 aactaatggt aaactgacgc tgaagttcat ctgtactact ggtaaactgc cggttccttg
     1501 gccgactctg gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt atccggacca
     1561 tatgaagcag catgacttct tcaagtccgc catgccggaa ggctatgtgc aggaacgcac
     1621 gatttccttt aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga
     1681 taccctggta aaccgcattg agctgaaagg cattgacttt aaagaggacg gcaatatcct
     1741 gggccataag ctggaataca attttaacag ccacaatgtt tacatcaccg ccgataaaca
     1801 aaaaaatggc attaaagcga attttaaaat tcgccacaac gtggaggatg gcagcgtgca
     1861 gctggctgat cactaccagc aaaacactcc aatcggtgat ggtcctgttc tgctgccaga
     1921 caatcactat ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga aacgcgatca
     1981 tatggttctg ctggagttcg taaccgcagc gggcatcacg catggtatgg atgaactgta
     2041 caaatgacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     2101 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     2161 cctttctgcg tttataggtc tcagctggaa atctgctcgt cagtggtgct cacactgacg
     2221 aatcatgtac agatcatacc gatgactgcc tggcgactca caactaagca agacagccgg
     2281 aaccagcgcc ggcgaacacc actgcatata tggcatatca caacagtcca cgtctcaagc
     2341 agttacagag atgttacgaa ccactagtgc actgcagtac aactagagat ctatgtgagg
     2401 atcctaactc gagatcgaga gtcctgcagg actagtgctg aggcattaat acatgggaag
     2461 attcgctttt tttttttgaa ttacaatagt atgtctgatg tctgcaagaa gtaacaggcg
     2521 tgtgcacaag aatacgtgtg tgtgcgtaag cgtatgcact ggtggcataa cttatctaag
     2581 aagtatatat cactgacata gaaatgtaga tatacaggta tttttctcga taatcgataa
     2641 aaatctcgtc gcgctgaacc aaacttggtg gttacggaga gtttttctct catcattact
     2701 gtctttcgca ttgatttccc ctttgaccga taaaatccct tggattcata agattaaaca
     2761 aagaggtgat caaagagaac cctgtgaaag tttatgttta taaccgggca taaagtgaac
     2821 tagacacttt caagaagcca accaaagcat gagtaacgaa gcttaccagc atgatcatac
     2881 cgtaaatcct caccagatta agacctcagc gccgagcggc cgcgtgttac aaccaattaa
     2941 ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     3001 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     3061 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3121 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3181 gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt
     3241 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3301 ttcgtgattg cgcctgagcg aggcgaaata cgcgatcgct gttaaaagga caattacaaa
     3361 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3421 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3481 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3541 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3601 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3661 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3721 ttaatcgcgg cctggagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3781 tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa
     3841 tgtaacatca gagattttga gacacaacgt ggctttgttg aataaatcga acttttgctg
     3901 agttgaagga tcagtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3961 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4021 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4081 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4141 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4201 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4261 gttggactc
//
