LOCUS       sequence-353631-        4337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  preassembled yeast MoClo level-2 backbone with GFP dropout for 
            genomic integration in site XI-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4337)
  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 4337)
  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-XI1
FEATURES             Location/Qualifiers
     source          1..4337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..85
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      299..887
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     788..807
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1829..1840
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1847..2560
                     /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      2572..2643
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2659..2686
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(3596..4337,1..74))
                     /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(join(3596..4337,1..74))
                     /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     join(4319..4337,1)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ccatataaat cagcatccat gttggaattt aatcgcggcc tggagcaaga cgtttcccgt
       61 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
      121 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg
      181 ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc agtcatgacc aaaatccctt
      241 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
      301 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
      361 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
      421 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
      481 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
      541 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
      601 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
      661 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
      721 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
      781 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
      841 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
      901 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      961 tatcccctga ttctgtggat aaccgtgcgg ccgccaatgc gctgagggtt taatggaata
     1021 gtgacgttgt gatgcggtga gttcggcggt taggggaatg gtatatgata aaaaacggaa
     1081 acgtgcttct ttaatttaat tgtttaatat tgttgcagat atataaaaag ggggaaagaa
     1141 ccgaagatgt aattattttt ttatcgcctc aacctaaagc aagcaataag gtataaagat
     1201 caggacgact cgagcgctga tatctaaatt tgaagccacg caagtaacta cgtaggtcag
     1261 aggggacaag gaataacact tgacattttt cttttttctt tttttttctt tttttttttt
     1321 ttgttaatct tggcttctgt accgtagcct cctgtatacg gttttagatg cagtatgttt
     1381 atcatcgccg agaaatttgg ggggtgcaaa ggaataaaat ccgtgctgaa atccgtgcac
     1441 cgcatcaaac tttctcggag gattctttgc agccgattaa gtcctcagcg agctcgccct
     1501 gaattcgcat ctagactgat gagacgtggt agagccacaa acagccggta caagcaacga
     1561 tctccaggac catctgaatc atgcgcggat gacacgaact cacgacggcg atcacagaca
     1621 ttaacccaca gtacagacac tgcgacaacg tggcaattcg tcgcaataca acgtgagacc
     1681 gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt gtaaatctta tttaataatg
     1741 tgtgcggcaa ttcacattta atttatgaat gttttcttaa catcgcggca actcaagaaa
     1801 cggcaggttc ggatcttagc tactagagaa agaggagaaa tactagatgc gtaaaggcga
     1861 agagctgttc actggtgtcg tccctattct ggtggaactg gatggtgatg tcaacggtca
     1921 taagttttcc gtgcgtggcg agggtgaagg tgacgcaact aatggtaaac tgacgctgaa
     1981 gttcatctgt actactggta aactgccggt tccttggccg actctggtaa cgacgctgac
     2041 ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg aagcagcatg acttcttcaa
     2101 gtccgccatg ccggaaggct atgtgcagga acgcacgatt tcctttaagg atgacggcac
     2161 gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc ctggtaaacc gcattgagct
     2221 gaaaggcatt gactttaaag aggacggcaa tatcctgggc cataagctgg aatacaattt
     2281 taacagccac aatgtttaca tcaccgccga taaacaaaaa aatggcatta aagcgaattt
     2341 taaaattcgc cacaacgtgg aggatggcag cgtgcagctg gctgatcact accagcaaaa
     2401 cactccaatc ggtgatggtc ctgttctgct gccagacaat cactatctga gcacgcaaag
     2461 cgttctgtct aaagatccga acgagaaacg cgatcatatg gttctgctgg agttcgtaac
     2521 cgcagcgggc atcacgcatg gtatggatga actgtacaaa tgaccaggca tcaaataaaa
     2581 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     2641 ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta taggtctcag
     2701 ctggaaatct gctcgtcagt ggtgctcaca ctgacgaatc atgtacagat cataccgatg
     2761 actgcctggc gactcacaac taagcaagac agccggaacc agcgccggcg aacaccactg
     2821 catatatggc atatcacaac agtccacgtc tcaagcagtt acagagatgt tacgaaccac
     2881 tagtgcactg cagtacaact agagatctat gtgaggatcc taactcgaga tcgagagtcc
     2941 tgcaggacta gtgctgaggc attaatagtt tcttggcatt ggcaaatctc tgctaaatgc
     3001 tgcgtacaga cggaaactca caccgccgcg aaaactggtc agtggcaaaa aaaaaaaaat
     3061 taaaaaaata aaaaataact attacgtatg atactgtttc tggtagttga tatgaggttg
     3121 gtgttgtata ttgtacgttt taggaacagg gaagtgaata ttatttactc tgctgcacat
     3181 tctggctagg tcgaagccgg aacctgagat gacgccgcgc tagaactatg gaccaagctg
     3241 ttgacaatgt ttagatggtg atgcactacc ctgtgcgggg agtggccacg aacgcgagcg
     3301 gaaggtgcgg gtgttgcggg aattgcggga ggttcttcgc taagcgtgag ggttgctagt
     3361 tggggcggcg gggtttccct aagtgtaaat agggcctcgc cgctggcaca tgagtgcgcc
     3421 ggaggaggcg gcggaggcga cgacgctaaa accgtggccg ttggggaagg atgggcggct
     3481 atatctacca ttgacctgat ggggactcgg ctcttaagga atgggtttga ggtgggtgtg
     3541 gattaagacc tcagcgccga gcggccgcgt gttacaacca attaaccaat tctgattaga
     3601 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     3661 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     3721 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     3781 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     3841 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat
     3901 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     3961 gagcgaggcg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     4021 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     4081 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     4141 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     4201 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     4261 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     4321 cgcgagccca tttatac
//
