LOCUS       sequence-431068-        6340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration backbone for the LYS2 integration site of S. cerevisiae.
            Includes a Leu2 nutritional marker and a sfGFP drop-out..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6340)
  AUTHORS   Martinusen SG, Slaton EW, Nelson SE, Pulgar MA, Besu JT, Simas CF, 
            Denard CA
  TITLE     Modular and integrative activity reporters enhance biochemical 
            studies in the yeast ER.
  JOURNAL   Protein Eng Des Sel. 2024 Jan 29;37:gzae008. doi: 
            10.1093/protein/gzae008.
  PUBMED    38696722
REFERENCE   2  (bases 1 to 6340)
  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"; journalName: "Protein Eng
            Des Sel."; date: "2024-01-29"; volume: "37:gzae008. doi"; pages: " 
            10.1093/protein/gzae008"
COMMENT     Sequence Label: LYS-Leu2
FEATURES             Location/Qualifiers
     source          1..6340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(59..86)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(102..173)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(185..898)
                     /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"
     RBS             905..916
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     rep_origin      complement(1981..2569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2061..2080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2783..2802)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2794..3609
                     /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             2794..3609
                     /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     complement(2867..2886)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(4466..5560)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(5561..5968)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 cagtgtgagc accactgacg agcagatttc cgtcgcatgc tcagctgaga cctataaacg
       61 cagaaaggcc cacccgaagg tgagccagtg tgactctagt agagagcgtt caccgacaaa
      121 caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctg
      181 gtcatttgta cagttcatcc ataccatgcg tgatgcccgc tgcggttacg aactccagca
      241 gaaccatatg atcgcgtttc tcgttcggat ctttagacag aacgctttgc gtgctcagat
      301 agtgattgtc tggcagcaga acaggaccat caccgattgg agtgttttgc tggtagtgat
      361 cagccagctg cacgctgcca tcctccacgt tgtggcgaat tttaaaattc gctttaatgc
      421 catttttttg tttatcggcg gtgatgtaaa cattgtggct gttaaaattg tattccagct
      481 tatggcccag gatattgccg tcctctttaa agtcaatgcc tttcagctca atgcggttta
      541 ccagggtatc gccttcaaat ttcacttccg cacgcgtttt gtacgtgccg tcatccttaa
      601 aggaaatcgt gcgttcctgc acatagcctt ccggcatggc ggacttgaag aagtcatgct
      661 gcttcatatg gtccggataa cgagcaaagc actgaacacc ataagtcagc gtcgttacca
      721 gagtcggcca aggaaccggc agtttaccag tagtacagat gaacttcagc gtcagtttac
      781 cattagttgc gtcaccttca ccctcgccac gcacggaaaa cttatgaccg ttgacatcac
      841 catccagttc caccagaata gggacgacac cagtgaacag ctcttcgcct ttacgcatct
      901 agtatttctc ctctttctct agtagctaag atccgaacct gccgtttctt gagttgccgc
      961 gatgttaaga aaacattcat aaattaaatg tgaattgccg cacacattat taaataagat
     1021 ttacaaaatg ttcaaaatga cgcatgaaat cacgtttcac tttcggtctc acgttcgtct
     1081 gcaattatcg gccagttcag tgagatggta ttgcgacgaa ttgccacgtt gtcgcagtgt
     1141 ctgtactgtg ggttaatgtc tgtgatcgcc gtcgtgagtt cgtgtcatcc gcgcatgatt
     1201 cagatggtcc tggagatcgt tgcttgtacc ggctgtttgt ggctctacca tctagatgcg
     1261 aattcaggga gcagtcaaat attgagtaaa ttggtccgca acaatggtta ctctttcatt
     1321 cgaatacagt aagctgttat aaattaagtt caaaacatgc gcattattac tggtatccaa
     1381 attcaaagca aagtccacta aatgatgctt gaactcgtct aatttgaaat cttggttttc
     1441 caaaaaggcc aaacggaaca actgaggggt cctttccaga tcttggcaac tttgaatttt
     1501 ttcagctagc tcgtcaaagg aaaaattggc ctcaatagaa ttgagcttgt tcaactcatt
     1561 gttaattgta gaatacagct cattaaatga ccacgttggt tgaatattga atcttaagat
     1621 tttgttattc gcaatataaa gaacaatatc atcgtcaccg gttactctat atatcaatgc
     1681 agcccataca ctcaaagcga cagcgtattt gttagaaaaa ctatcatgag gcacatcgag
     1741 ctgaggtagc tgtaacgaat atgtagcttg tttcgtataa ggttcttgtt gtgggcgtaa
     1801 aaagtcatgt ggtaacactg aaagagttgg attatccaac ttctctatcc agaccttttc
     1861 gttagtcata ttggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1921 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1981 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2041 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2101 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2161 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2221 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2281 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2341 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2401 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2461 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2521 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2581 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2641 catgactgat ccttcaactc agcaaaagtt cgatttattc aacaaagcca cgttgtgtct
     2701 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     2761 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     2821 tgctccaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     2881 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     2941 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     3001 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     3061 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     3121 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     3181 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgcctc
     3241 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     3301 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca
     3361 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     3421 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     3481 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     3541 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     3601 tttttctaat cagaattggt taattggttg taacacgcgg ccgctcggtt aagctgctgc
     3661 ggagcttcca cgagcaccag cacctgaagc aactagactt atttgcgctt gagttagttc
     3721 tatacttggc agtggaagtt tgtcattatg agttggtgga ggtaaaaatc caaccttgtt
     3781 taaaaatgca atatatatac caacctcttc tggagtaaca cctattccat tagaccaatc
     3841 aacacctgtc catgcggtgt ctttctttaa agatgccacg gcgttcctat cgtctagttc
     3901 cggagctttg gtactttcag gtaagttgtc taagaccatg tgtagcaaag gatacaacgc
     3961 attttcttca ttcctgtcaa taacagacgc ctccaatgat ttcttccatt tagaatagct
     4021 ttcgatttcg acatcgtaac cataatcgtg taaagtatac aagtagtctt tgaataatat
     4081 tcttgggtga cccgttactt gagcaacggc caattcattt tctttgggag gattcaaaga
     4141 cgtagcaaca acaacacgag caacatgatc tactggaacc atattcacgg aattttcgat
     4201 atctggaatc ttacctaatt ggactgaacc tttcaaaaat ctcaataaga aatcatctgt
     4261 gtttgaagaa ccattggcag aggcaccact cctgcctatt taacgccaac gttgcgagat
     4321 atacactcga aatgtgtgat gtagatgtaa acagacggct atatgaatgc cactagccgg
     4381 ttattttact cctagttaat acatatttcc tagaattttt tacgtgcata agagtagaat
     4441 agtctaaaga gaagggtcct ccaggttaag caaggatttt cttaacttct tcggcgacag
     4501 catcaccgac ttcggtggta ctgttggaac cacctaaatc accagttctg atacctgcat
     4561 ccaaaacctt tttaactgca tcttcaatgg ccttaccttc ttcaggcaag ttcaatgaca
     4621 atttcaacat cattgcagca gacaagatag tggcgatagg gttgacctta ttctttggca
     4681 aatctggagc agaaccgtgg catggttcgt acaaaccaaa tgcggtgttc ttgtctggca
     4741 aagaggccaa ggacgcagat ggcaacaaac ccaaggaacc tgggataacg gaggcttcat
     4801 cggagatgat atcaccaaac atgttgctgg tgattataat accatttagg tgggttgggt
     4861 tcttaactag gatcatggcg gcagaatcaa tcaattgatg ttgaaccttc aatgtaggaa
     4921 attcgttctt gatggtttcc tccacagttt ttctccataa tcttgaagag gccaaaacat
     4981 tagctttatc caaggaccaa ataggcaatg gtggctcatg ttgtagggcc atgaaagcgg
     5041 ccattcttgt gattctttgc acttctggaa cggtgtattg ttcactatcc caagcgacac
     5101 catcaccatc gtcctccttt ctcttaccaa agtaaatacc tcccactaat tctctgacaa
     5161 caacgaagtc agtaccttta gcaaattgtg gcttgattgg agataagtct aaaagagagt
     5221 cggatgcaaa gttacatggt cttaagttgg cgtacaattg aagttcttta cggattttta
     5281 gtaaaccttg ttcaggtcta acactaccgg ttccccattt aggaccaccc acagcaccta
     5341 acaaaacggc atcagccttc ttggaggctt ccagcgcctc atctggaagt ggaacacctg
     5401 tagcatcgat agcagcacca ccaattaaat gattttcgaa atcgaacttg acattggaac
     5461 gaacatcaga aatagcttta agaaccttaa tggcttcggc tgtgatttct tgaccaacgt
     5521 ggtcacctgg caaaacgacg atcttcttag gggcagacat tagaatggta tatccttgaa
     5581 atatatatat atatattgct gaaatgtaaa aggtaagaaa agttagaaag taagacgatt
     5641 gctaaccacc tattggaaaa aacaataggt ccttaaataa tattgtcaac ttcaagtatt
     5701 gtgatgcaag catttagtca tgaacgcttc tctattctat atgaaaagcc ggttccggcg
     5761 ctctcacctt tcctttttct cccaattttt cagttgaaaa aggtatatgc gtcaggcgac
     5821 ctctgaaatt aacaaaaaat ttccagtcat cgaatttgat tctgtgcgat agcgcccctg
     5881 tgtgttctcg ttatgttgag gaaaaaaata atggttgcta agagattcga actcttgcat
     5941 cttacgatac ctgagtattc ccacagttaa ctgcggtcaa gatatttctt gaatcaggcg
     6001 ccttagaccg ctcggccaaa caaccaatta cttgttgaga aatagagtat aattatccta
     6061 taaatataac gtttttgaac acacatgaac aaggaagtac aggacaattg attttgaaga
     6121 gaatgtggat tttgatgtaa ttgttgggat tccattttta ataaggcaat aatattaggt
     6181 atgtagatat actagaagtt ctcctcgatg tactgcagtg cactagttgg actgttgtga
     6241 tatgccatat atgcagtggt gttcgccggc gctggttccg gctgtcttgc ttagttgtga
     6301 gtcgccaggc agtcatcggt atgatctgta catgattcgt
//
