LOCUS       sequence-346554-        4404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site XII-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4404)
  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 4404)
  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-XII1
FEATURES             Location/Qualifiers
     source          1..4404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(522..1337)
                     /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(522..1337)
                     /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     1245..1264
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1329..1348
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1562..2150
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2051..2070
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             3145..3156
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3163..3876
                     /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      3888..3959
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3975..4002
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 aatactaatt agtctttgcc ggttacccat ctggcccctg acttgcgatg cttaggaagt
       61 tccatactcg cggctcttcc caacagtagc acatccgtga aacttctggc gctattcatt
      121 atgcagtacc aggacaagaa gttaaaaaaa aagctctgtt acaagttcaa tggtggtgca
      181 aggattgaag ttattatcca ggaggcacgg atgcaaaaga tagaggacaa attaatttcc
      241 ttaaaattca aaatgctcat tattgtcacg ctgtatgagc atttggtgaa gatttcactg
      301 ggaaatgttg caataatttg ataatcgttc gtattggatg aaactgtaac atcatctgtt
      361 tattaagtat ccgtgttatt agtatatcat cacatacggt gtaagaagat aacataaaga
      421 ttgagaaaca gtcatcaaat ataatggaag ctgaaatgcg aggattgatt aagacctcag
      481 cgccgagcgg ccgcgtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc
      541 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
      601 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
      661 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
      721 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
      781 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
      841 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gaggcgaaat
      901 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
      961 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     1021 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     1081 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1141 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     1201 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     1261 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca agacgtttcc
     1321 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     1381 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg
     1441 tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagtcatg accaaaatcc
     1501 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     1561 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     1621 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     1681 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     1741 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     1801 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     1861 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     1921 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     1981 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2041 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2101 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2161 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     2221 cgttatcccc tgattctgtg gataaccgtg cggccgccaa tgcgctgagg gtttaatgtt
     2281 gagctctgtc cttcatggac tttttggacc acttcttctt ggacttctta ccaccagcaa
     2341 gagcggcagc ggctttggca gctttagaca attgttgctt tggaggcatg ttatattatg
     2401 ttctgagtcg taagaaaggg tgaaaattga tgttagtgtc aaaaattata ttacaaaata
     2461 cgcagagata ttctagttcc tttgatgaat gaatctttca gaaaaaaaag tcaaagcaaa
     2521 agcaaaatgg cctgcagact aaactgtatg gtggtcttgg aatgataaag atctgtttaa
     2581 tagatttagt agatacaata gcacatctca ttacccagtt atgattgacg tcattctgag
     2641 ttacaatgat cttagcgacc tgtgctcatt atttgctcca ctaattctaa ttttcctcgc
     2701 ctttcatatt tcgtatcttt attctatatc ctaaaatttt tttggcaaat cccagatttg
     2761 gctttgattt tggcatcggt tcggttcttt cattaagtcc tcagcgagct cgccctgaat
     2821 tcgcatctag actgatgaga cgtggtagag ccacaaacag ccggtacaag caacgatctc
     2881 caggaccatc tgaatcatgc gcggatgaca cgaactcacg acggcgatca cagacattaa
     2941 cccacagtac agacactgcg acaacgtggc aattcgtcgc aatacaacgt gagaccgaaa
     3001 gtgaaacgtg atttcatgcg tcattttgaa cattttgtaa atcttattta ataatgtgtg
     3061 cggcaattca catttaattt atgaatgttt tcttaacatc gcggcaactc aagaaacggc
     3121 aggttcggat cttagctact agagaaagag gagaaatact agatgcgtaa aggcgaagag
     3181 ctgttcactg gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa cggtcataag
     3241 ttttccgtgc gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac gctgaagttc
     3301 atctgtacta ctggtaaact gccggttcct tggccgactc tggtaacgac gctgacttat
     3361 ggtgttcagt gctttgctcg ttatccggac catatgaagc agcatgactt cttcaagtcc
     3421 gccatgccgg aaggctatgt gcaggaacgc acgatttcct ttaaggatga cggcacgtac
     3481 aaaacgcgtg cggaagtgaa atttgaaggc gataccctgg taaaccgcat tgagctgaaa
     3541 ggcattgact ttaaagagga cggcaatatc ctgggccata agctggaata caattttaac
     3601 agccacaatg tttacatcac cgccgataaa caaaaaaatg gcattaaagc gaattttaaa
     3661 attcgccaca acgtggagga tggcagcgtg cagctggctg atcactacca gcaaaacact
     3721 ccaatcggtg atggtcctgt tctgctgcca gacaatcact atctgagcac gcaaagcgtt
     3781 ctgtctaaag atccgaacga gaaacgcgat catatggttc tgctggagtt cgtaaccgca
     3841 gcgggcatca cgcatggtat ggatgaactg tacaaatgac caggcatcaa ataaaacgaa
     3901 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct
     3961 actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatagg tctcagctgg
     4021 aaatctgctc gtcagtggtg ctcacactga cgaatcatgt acagatcata ccgatgactg
     4081 cctggcgact cacaactaag caagacagcc ggaaccagcg ccggcgaaca ccactgcata
     4141 tatggcatat cacaacagtc cacgtctcaa gcagttacag agatgttacg aaccactagt
     4201 gcactgcagt acaactagag atctatgtga ggatcctaac tcgagatcga gagtcctgca
     4261 ggactagtgc tgaggcatta atcttcccgt gaatcaactg cacatacctt aataactagt
     4321 tcagtttagt gctctgtctg agtgactgta ataataaatt ttacaagcca cttctcatga
     4381 catatattgg taagtaactt catc
//
