LOCUS       sequence-346545-        4433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site XI-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4433)
  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 4433)
  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-XI2
FEATURES             Location/Qualifiers
     source          1..4433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(36..851)
                     /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(36..851)
                     /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     759..778
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     843..862
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1076..1664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1565..1584
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             2714..2725
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2732..3445
                     /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      3457..3528
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3544..3571
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 gcggccgcgt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
       61 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
      121 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
      181 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
      241 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc
      301 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
      361 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgaggcg aaatacgcga
      421 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
      481 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
      541 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
      601 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
      661 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
      721 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
      781 tataaatcag catccatgtt ggaatttaat cgcggcctgg agcaagacgt ttcccgttga
      841 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
      901 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt
      961 tgttgaataa atcgaacttt tgctgagttg aaggatcagt catgaccaaa atcccttaac
     1021 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1081 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1141 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1201 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     1261 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1321 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1381 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1441 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1501 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1561 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     1621 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     1681 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     1741 cccctgattc tgtggataac cgtgcggccg ccaatgcgct gagggtttaa tgaggatttt
     1801 cgatggagca ggatgaggag aaatagtacc acatgtatat atccattaca aaaaggttta
     1861 tatacaatta caatagaccc ttgttggggt ttctgaaaaa agaagtagtc gatgccatcg
     1921 gcaataatac ggaattacga gaaacacaat cccgatcctt ttttgggtaa ttacttcacc
     1981 gattctaccg atttatcatg ccaaaaaaaa ttcaccgtgg gttctagaag tgccctttga
     2041 ggattgtagc cactctaacc cacacggcct ccttactagc tgactaaggt gacaaaaccg
     2101 caaggactgg aaagtcgcca ctcatctgaa aattctcaag tttttcacta ctgagtttat
     2161 gctttcgaat ttttttgttc ggtaatagca cggcggttcg attcaattcc gccgctccga
     2221 gcgatgctcc gcaaaactca gtaataagct ttctgatggt tcaccccttt tttagcacgc
     2281 ggggtgtaac tcaacagaaa aatgtgccat agaacaagac taggcaaaag ccaaggagcg
     2341 tttgccatga acttccacaa attaagtcct cagcgagctc gccctgaatt cgcatctaga
     2401 ctgatgagac gtggtagagc cacaaacagc cggtacaagc aacgatctcc aggaccatct
     2461 gaatcatgcg cggatgacac gaactcacga cggcgatcac agacattaac ccacagtaca
     2521 gacactgcga caacgtggca attcgtcgca atacaacgtg agaccgaaag tgaaacgtga
     2581 tttcatgcgt cattttgaac attttgtaaa tcttatttaa taatgtgtgc ggcaattcac
     2641 atttaattta tgaatgtttt cttaacatcg cggcaactca agaaacggca ggttcggatc
     2701 ttagctacta gagaaagagg agaaatacta gatgcgtaaa ggcgaagagc tgttcactgg
     2761 tgtcgtccct attctggtgg aactggatgg tgatgtcaac ggtcataagt tttccgtgcg
     2821 tggcgagggt gaaggtgacg caactaatgg taaactgacg ctgaagttca tctgtactac
     2881 tggtaaactg ccggttcctt ggccgactct ggtaacgacg ctgacttatg gtgttcagtg
     2941 ctttgctcgt tatccggacc atatgaagca gcatgacttc ttcaagtccg ccatgccgga
     3001 aggctatgtg caggaacgca cgatttcctt taaggatgac ggcacgtaca aaacgcgtgc
     3061 ggaagtgaaa tttgaaggcg ataccctggt aaaccgcatt gagctgaaag gcattgactt
     3121 taaagaggac ggcaatatcc tgggccataa gctggaatac aattttaaca gccacaatgt
     3181 ttacatcacc gccgataaac aaaaaaatgg cattaaagcg aattttaaaa ttcgccacaa
     3241 cgtggaggat ggcagcgtgc agctggctga tcactaccag caaaacactc caatcggtga
     3301 tggtcctgtt ctgctgccag acaatcacta tctgagcacg caaagcgttc tgtctaaaga
     3361 tccgaacgag aaacgcgatc atatggttct gctggagttc gtaaccgcag cgggcatcac
     3421 gcatggtatg gatgaactgt acaaatgacc aggcatcaaa taaaacgaaa ggctcagtcg
     3481 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac
     3541 actggctcac cttcgggtgg gcctttctgc gtttataggt ctcagctgga aatctgctcg
     3601 tcagtggtgc tcacactgac gaatcatgta cagatcatac cgatgactgc ctggcgactc
     3661 acaactaagc aagacagccg gaaccagcgc cggcgaacac cactgcatat atggcatatc
     3721 acaacagtcc acgtctcaag cagttacaga gatgttacga accactagtg cactgcagta
     3781 caactagaga tctatgtgag gatcctaact cgagatcgag agtcctgcag gactagtgct
     3841 gaggcattaa tcacacgact agcgctttca gatattaaaa agtttagatg taggttttag
     3901 cggtaacagt tatataaatc gtgtttcttc tcttgatgaa acaaaaaaat gctagaaaaa
     3961 ctttgtcgtt tcttactttt ggtgcgcttt gcagttttcg tggctagact tagaatcatt
     4021 tctcctcaga tttcttgatt aaagtttggt gcgaagccct actctaacat tggtgttctt
     4081 cttttcattc acgcaagtta agtccaggaa ggtgagcaaa tgctcatcct tctgttcatg
     4141 cgtgacggct gaattatcct tatctggcgt acccgtgcag ccgtttccgt gcctcggttc
     4201 ctccgagata tccttaggga ccgccaggga ccatgattgc gtcaactgtt gtcaccgctc
     4261 cagaggatcc tctgtaacct tttcaaccat aaaaatagaa agggcacagg gttggggtat
     4321 ttgaattttt tttttgggtt ttttcgtatt acttattact taccttcttt tctatataat
     4381 tttgtttttc cctggtagag cggaatcttc ccacattaag acctcagcgc cga
//
