LOCUS       sequence-496006-        6340 bp    DNA     circular SYN 23-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 23, 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"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1654..1665
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1672..2385
                     /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      2397..2468
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2484..2511
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2942..3349
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3350..4444
                     /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"
     CDS             complement(5301..6116)
                     /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(5301..6116)
                     /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     6024..6043
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6108..6127
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtgc ggccgccaat atgactaacg aaaaggtctg
      721 gatagagaag ttggataatc caactctttc agtgttacca catgactttt tacgcccaca
      781 acaagaacct tatacgaaac aagctacata ttcgttacag ctacctcagc tcgatgtgcc
      841 tcatgatagt ttttctaaca aatacgctgt cgctttgagt gtatgggctg cattgatata
      901 tagagtaacc ggtgacgatg atattgttct ttatattgcg aataacaaaa tcttaagatt
      961 caatattcaa ccaacgtggt catttaatga gctgtattct acaattaaca atgagttgaa
     1021 caagctcaat tctattgagg ccaatttttc ctttgacgag ctagctgaaa aaattcaaag
     1081 ttgccaagat ctggaaagga cccctcagtt gttccgtttg gcctttttgg aaaaccaaga
     1141 tttcaaatta gacgagttca agcatcattt agtggacttt gctttgaatt tggataccag
     1201 taataatgcg catgttttga acttaattta taacagctta ctgtattcga atgaaagagt
     1261 aaccattgtt gcggaccaat ttactcaata tttgactgct ccctgaattc gcatctagat
     1321 ggtagagcca caaacagccg gtacaagcaa cgatctccag gaccatctga atcatgcgcg
     1381 gatgacacga actcacgacg gcgatcacag acattaaccc acagtacaga cactgcgaca
     1441 acgtggcaat tcgtcgcaat accatctcac tgaactggcc gataattgca gacgaacgtg
     1501 agaccgaaag tgaaacgtga tttcatgcgt cattttgaac attttgtaaa tcttatttaa
     1561 taatgtgtgc ggcaattcac atttaattta tgaatgtttt cttaacatcg cggcaactca
     1621 agaaacggca ggttcggatc ttagctacta gagaaagagg agaaatacta gatgcgtaaa
     1681 ggcgaagagc tgttcactgg tgtcgtccct attctggtgg aactggatgg tgatgtcaac
     1741 ggtcataagt tttccgtgcg tggcgagggt gaaggtgacg caactaatgg taaactgacg
     1801 ctgaagttca tctgtactac tggtaaactg ccggttcctt ggccgactct ggtaacgacg
     1861 ctgacttatg gtgttcagtg ctttgctcgt tatccggacc atatgaagca gcatgacttc
     1921 ttcaagtccg ccatgccgga aggctatgtg caggaacgca cgatttcctt taaggatgac
     1981 ggcacgtaca aaacgcgtgc ggaagtgaaa tttgaaggcg ataccctggt aaaccgcatt
     2041 gagctgaaag gcattgactt taaagaggac ggcaatatcc tgggccataa gctggaatac
     2101 aattttaaca gccacaatgt ttacatcacc gccgataaac aaaaaaatgg cattaaagcg
     2161 aattttaaaa ttcgccacaa cgtggaggat ggcagcgtgc agctggctga tcactaccag
     2221 caaaacactc caatcggtga tggtcctgtt ctgctgccag acaatcacta tctgagcacg
     2281 caaagcgttc tgtctaaaga tccgaacgag aaacgcgatc atatggttct gctggagttc
     2341 gtaaccgcag cgggcatcac gcatggtatg gatgaactgt acaaatgacc aggcatcaaa
     2401 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     2461 acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttataggt
     2521 ctcagctgag catgcgacgg aaatctgctc gtcagtggtg ctcacactga cgaatcatgt
     2581 acagatcata ccgatgactg cctggcgact cacaactaag caagacagcc ggaaccagcg
     2641 ccggcgaaca ccactgcata tatggcatat cacaacagtc caactagtgc actgcagtac
     2701 atcgaggaga acttctagta tatctacata cctaatatta ttgccttatt aaaaatggaa
     2761 tcccaacaat tacatcaaaa tccacattct cttcaaaatc aattgtcctg tacttccttg
     2821 ttcatgtgtg ttcaaaaacg ttatatttat aggataatta tactctattt ctcaacaagt
     2881 aattggttgt ttggccgagc ggtctaaggc gcctgattca agaaatatct tgaccgcagt
     2941 taactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt agcaaccatt
     3001 atttttttcc tcaacataac gagaacacac aggggcgcta tcgcacagaa tcaaattcga
     3061 tgactggaaa ttttttgtta atttcagagg tcgcctgacg catatacctt tttcaactga
     3121 aaaattggga gaaaaaggaa aggtgagagc gccggaaccg gcttttcata tagaatagag
     3181 aagcgttcat gactaaatgc ttgcatcaca atacttgaag ttgacaatat tatttaagga
     3241 cctattgttt tttccaatag gtggttagca atcgtcttac tttctaactt ttcttacctt
     3301 ttacatttca gcaatatata tatatatatt tcaaggatat accattctaa tgtctgcccc
     3361 taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag ccgaagccat
     3421 taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt tcgaaaatca
     3481 tttaattggt ggtgctgcta tcgatgctac aggtgttcca cttccagatg aggcgctgga
     3541 agcctccaag aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta aatggggaac
     3601 cggtagtgtt agacctgaac aaggtttact aaaaatccgt aaagaacttc aattgtacgc
     3661 caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc caatcaagcc
     3721 acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag gtatttactt
     3781 tggtaagaga aaggaggacg atggtgatgg tgtcgcttgg gatagtgaac aatacaccgt
     3841 tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac atgagccacc
     3901 attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat tatggagaaa
     3961 aactgtggag gaaaccatca agaacgaatt tcctacattg aaggttcaac atcaattgat
     4021 tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta ttataatcac
     4081 cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag gttccttggg
     4141 tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat ttggtttgta
     4201 cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggtcaacc ctatcgccac
     4261 tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag aaggtaaggc
     4321 cattgaagat gcagttaaaa aggttttgga tgcaggtatc agaactggtg atttaggtgg
     4381 ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga aaatccttgc
     4441 ttaacctgga ggacccttct ctttagacta ttctactctt atgcacgtaa aaaattctag
     4501 gaaatatgta ttaactagga gtaaaataac cggctagtgg cattcatata gccgtctgtt
     4561 tacatctaca tcacacattt cgagtgtata tctcgcaacg ttggcgttaa ataggcagga
     4621 gtggtgcctc tgccaatggt tcttcaaaca cagatgattt cttattgaga tttttgaaag
     4681 gttcagtcca attaggtaag attccagata tcgaaaattc cgtgaatatg gttccagtag
     4741 atcatgttgc tcgtgttgtt gttgctacgt ctttgaatcc tcccaaagaa aatgaattgg
     4801 ccgttgctca agtaacgggt cacccaagaa tattattcaa agactacttg tatactttac
     4861 acgattatgg ttacgatgtc gaaatcgaaa gctattctaa atggaagaaa tcattggagg
     4921 cgtctgttat tgacaggaat gaagaaaatg cgttgtatcc tttgctacac atggtcttag
     4981 acaacttacc tgaaagtacc aaagctccgg aactagacga taggaacgcc gtggcatctt
     5041 taaagaaaga caccgcatgg acaggtgttg attggtctaa tggaataggt gttactccag
     5101 aagaggttgg tatatatatt gcatttttaa acaaggttgg atttttacct ccaccaactc
     5161 ataatgacaa acttccactg ccaagtatag aactaactca agcgcaaata agtctagttg
     5221 cttcaggtgc tggtgctcgt ggaagctccg cagcagctta accgagcggc cgcgtgttac
     5281 aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     5341 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     5401 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     5461 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     5521 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc
     5581 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     5641 ccgttattca ttcgtgattg cgcctgagcg aggcgaaata cgcgatcgct gttaaaagga
     5701 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     5761 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     5821 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     5881 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     5941 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     6001 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     6061 atgttggaat ttaatcgcgg cctggagcaa gacgtttccc gttgaatatg gctcataaca
     6121 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta
     6181 tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttgttg aataaatcga
     6241 acttttgctg agttgaagga tcagtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6301 actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
