LOCUS       sequence-346543-        4419 bp    DNA     circular SYN 14-APR-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site XI-5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4419)
  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 4419)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-XI5
FEATURES             Location/Qualifiers
     source          1..4419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      392..463
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      479..506
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1296..2111)
                     /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(1296..2111)
                     /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     2019..2038
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2103..2122
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2336..2924
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2825..2844
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             4068..4079
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4086..4419,1..380)
                     /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"
ORIGIN
        1 tgaaatttga aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag
       61 aggacggcaa tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca
      121 tcaccgccga taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg
      181 aggatggcag cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc
      241 ctgttctgct gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga
      301 acgagaaacg cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg
      361 gtatggatga actgtacaaa tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga
      421 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg
      481 ctcaccttcg ggtgggcctt tctgcgttta taggtctcag ctggaaatct gctcgtcagt
      541 ggtgctcaca ctgacgaatc atgtacagat cataccgatg actgcctggc gactcacaac
      601 taagcaagac agccggaacc agcgccggcg aacaccactg catatatggc atatcacaac
      661 agtccacgtc tcaagcagtt acagagatgt tacgaaccac tagtgcactg cagtacaact
      721 agagatctat gtgaggatcc taactcgaga tcgagagtcc tgcaggacta gtgctgaggc
      781 attactattg gctgcttcat agtacaccca attgcccaca acccaagtta atcataagct
      841 cttgttgtcg atgaattgct caaaatgtgg ccattctttc aattttgcac ctctaaaact
      901 gctccgtggg ttgtgacaac aaccttaagg gtaatggact tttttagaaa gacaaatgag
      961 atgatttcac ctactggtat atcatttatt tatacacgac cgttttgaca tttcagagtt
     1021 gcacgttggt atatcctaat attctcacaa gcatcacagt gaagaaacgc aagaaaatgg
     1081 aaaaaattcc caggtggttg ttgtcttctc ttatatcgtc ggtgctgtgc atacttgggg
     1141 ccctgtgtgt gccgttgtta tcggttgcct tcgatagcaa gcgcaatagc caatctaagt
     1201 tggtcaacta tggtctttct ctaagtgccg gatctatgat cattaagacc tcagcgccga
     1261 gcggccgcgt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
     1321 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     1381 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     1441 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     1501 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc
     1561 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     1621 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgaggcg aaatacgcga
     1681 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     1741 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1801 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     1861 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1921 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1981 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     2041 tataaatcag catccatgtt ggaatttaat cgcggcctgg agcaagacgt ttcccgttga
     2101 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     2161 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt
     2221 tgttgaataa atcgaacttt tgctgagttg aaggatcagt catgaccaaa atcccttaac
     2281 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2341 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2401 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2461 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     2521 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2581 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2641 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2701 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2761 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2821 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2881 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2941 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3001 cccctgattc tgtggataac cgtgcggccg ccaatgcgct gagggtttaa tgcggagaag
     3061 tcgttgatag catttccgaa ggcttttcca cattgactgc agtatctaaa agagctccag
     3121 cagcggtctt agtagttcct gtgcttgccg ctttgcagta tacagggaca ttgggaacta
     3181 cggaggaaaa atcggaatgc tctgagctct tctcaaaacc agacgaactc gtcttatttc
     3241 tcagtaaatt acccaacata taaactctcc ctcttcacct tctctacaag gccacacaca
     3301 agacagtttt aacaggcaaa tggtactgga ggctctcacc aatggggttt aagacgctcc
     3361 aaacacacgc tctccttttt ttctttatat atcggtagca acgaaagcta gtcgcagaac
     3421 gttacacact aaagcagatc aatcaatcaa tcctgagaac ctgcttcgtt caaaccatcc
     3481 actagaagaa acacgcacgt gtacccaaaa atagagacaa acaaacagac aaaaataaca
     3541 atgattccta ccgactaaag taaaacaaca ctcacgtaat aaaaaatcct agatacaact
     3601 cacttgtatt cctctcttct ttgaccatca ctaaagcttc tctcttacta tattcctcaa
     3661 ataaaaaaaa gttgcaatga ttacttacca atgtgccata aactccgtgc accaattaag
     3721 tcctcagcga gctcgccctg aattcgcatc tagactgatg agacgtggta gagccacaaa
     3781 cagccggtac aagcaacgat ctccaggacc atctgaatca tgcgcggatg acacgaactc
     3841 acgacggcga tcacagacat taacccacag tacagacact gcgacaacgt ggcaattcgt
     3901 cgcaatacaa cgtgagaccg aaagtgaaac gtgatttcat gcgtcatttt gaacattttg
     3961 taaatcttat ttaataatgt gtgcggcaat tcacatttaa tttatgaatg ttttcttaac
     4021 atcgcggcaa ctcaagaaac ggcaggttcg gatcttagct actagagaaa gaggagaaat
     4081 actagatgcg taaaggcgaa gagctgttca ctggtgtcgt ccctattctg gtggaactgg
     4141 atggtgatgt caacggtcat aagttttccg tgcgtggcga gggtgaaggt gacgcaacta
     4201 atggtaaact gacgctgaag ttcatctgta ctactggtaa actgccggtt ccttggccga
     4261 ctctggtaac gacgctgact tatggtgttc agtgctttgc tcgttatccg gaccatatga
     4321 agcagcatga cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt
     4381 cctttaagga tgacggcacg tacaaaacgc gtgcggaag
//
