LOCUS       sequence-431067-        6276 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration backbone for the MET15 integration site of S. 
            cerevisiae. Includes a HygromycinR gene and a sfGFP drop-out..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6276)
  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 6276)
  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: MET-HYGRO
FEATURES             Location/Qualifiers
     source          1..6276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      812..1009
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1646..2461)
                     /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(1646..2461)
                     /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     2369..2388
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2453..2472
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2686..3274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3175..3194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             4624..4635
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4642..5355
                     /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
                     FICATGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      5367..5438
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5454..5481
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            join(5711..6276,1..1009)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     CDS             join(6055..6276,1..807)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWARGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
ORIGIN
        1 attccggaag tgcttgacat tggggaattt agcgagagcc tgacctattg catctcccgc
       61 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcaa
      121 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
      181 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
      241 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
      301 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
      361 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
      421 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
      481 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
      541 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
      601 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgcg gggtcgatgc
      661 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
      721 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
      781 agcactcgtc cgagggcaaa ggaataaagt aactgacaat aaaaagattc ttgttttcaa
      841 gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag
      901 cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca
      961 gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgg agtatgttgg
     1021 tgatgccaag accttagtca ttgctccata cttcactacc cacaaacaat taaatgacaa
     1081 agaaaagttg gcatctggtg ttaccaagga cttaattcgt gtctctgttg gtatcgaatt
     1141 tattgatgac attattgcag acttccagca atcttttgaa actgttttcg ctggccaaaa
     1201 accatgagtg tgcgtaatga gttgtaaaat tatgtataaa cctactttct ctcacaagta
     1261 ctatactttt ataaaacgaa ctttattgaa atgaatatcc tttttttccc ttgttacatg
     1321 tcgtgactcg tactttgaac ctaaattgtt ctaacatcaa agaacagtgt taattcgcag
     1381 tcgagaagaa aaatatggtg aacaagactc atctacttca tgagactact ttacgcctcc
     1441 tataaagctg tcacactgga taaatttatt gtaggaccaa gttacaatag aggatgatgg
     1501 aggtttcttt acaataaaga agcacatgtg tgttaacgtt tttagtattt gcttgttatg
     1561 taaatcagga aaacttcgcg ggatttggtt ggatgctact ttccatccga gcggccgcgt
     1621 gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca
     1681 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1741 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     1801 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1861 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt
     1921 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     1981 ccaaaccgtt attcattcgt gattgcgcct gagcgaggcg aaatacgcga tcgctgttaa
     2041 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     2101 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     2161 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     2221 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     2281 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     2341 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     2401 catccatgtt ggaatttaat cgcggcctgg agcaagacgt ttcccgttga atatggctca
     2461 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat
     2521 ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt tgttgaataa
     2581 atcgaacttt tgctgagttg aaggatcagt catgaccaaa atcccttaac gtgagttttc
     2641 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2701 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2761 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2821 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2881 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2941 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3001 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3061 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3121 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3181 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3241 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3301 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3361 tgtggataac cgtgcggccg ccaattcgag gatttaggaa tccataaaag ggaatctgca
     3421 attctacaca attctataaa tattattatc atcattttat atgttaatat tcattgatcc
     3481 tattacatta tcaatccttg cgtttcagct tccactaatt tagatgacta tttctcatca
     3541 tttgcgtcat cttctaacac cgtatatgat aatatactag taacgtaaat actagttagt
     3601 agatgatagt tgatttttat tccaacaaga tgactatttc tcatcatttg cgtcatcttc
     3661 taacaccgta tatgataata tactagtaac gtaaatacta gttagtagat gatagttgat
     3721 ttttattcca acactaagaa ataatttcgc catttcttga atgtatttaa agatatttaa
     3781 tgctataata gacatttaaa tccaattctt ccaacataca atgggagttt ggccgagtgg
     3841 tttaaggcgt cagatttagg tggatttaac ctctaaaatc tctgatatct tcggatgcaa
     3901 gggttcgaat cccttagctc tcattatttt ttgctttttc tcttgaggtc acatgatcgc
     3961 aaaatggcaa atggcacgtg aagctgtcga tattggggaa ctgtggtggt tggcaaatga
     4021 ctaattaagt tagtcaaggc gccatcctca tgaaaactgt gtaacataat aaccgaagtg
     4081 tcgaaaaggt ggcaccttgt ccaattgaac acgctcgatg aaaaaaataa gatatatata
     4141 aggttaagta aagcgtctgt tagaaaggaa gtttttcctt tttcttgctc tcttgtcttt
     4201 tcatctacta tttccttcgt gtaatacagg gtcgtcagat acatagatac aattctatta
     4261 cccccatcca ccctgaattc gcatctagat ggtagagcca caaacagccg gtacaagcaa
     4321 cgatctccag gaccatctga atcatgcgcg gatgacacga actcacgacg gcgatcacag
     4381 acattaaccc acagtacaga cactgcgaca acgtggcaat tcgtcgcaat accttctcac
     4441 tgaactggcc gataattgca gacgaacgtg agaccgaaag tgaaacgtga tttcatgcgt
     4501 cattttgaac attttgtaaa tcttatttaa taatgtgtgc ggcaattcac atttaattta
     4561 tgaatgtttt cttaacatcg cggcaactca agaaacggca ggttcggatc ttagctacta
     4621 gagaaagagg agaaatacta gatgcgtaaa ggcgaagagc tgttcactgg tgtcgtccct
     4681 attctggtgg aactggatgg tgatgtcaac ggtcataagt tttccgtgcg tggcgagggt
     4741 gaaggtgacg caactaatgg taaactgacg ctgaagttca tctgtgctac tggtaaactg
     4801 ccggttcctt ggccgactct ggtaacgacg ctgacttatg gtgttcagtg ctttgctcgt
     4861 tatccggacc atatgaagca gcatgacttc ttcaagtccg ccatgccgga aggctatgtg
     4921 caggaacgca cgatttcctt taaggatgac ggcacgtaca aaacgcgtgc ggaagtgaaa
     4981 tttgaaggcg ataccctggt aaaccgcatt gagctgaaag gcattgactt taaagaggac
     5041 ggcaatatcc tgggccataa gctggaatac aattttaaca gccacaatgt ttacatcacc
     5101 gccgataaac aaaaaaatgg cattaaagcg aattttaaaa ttcgccacaa cgtggaggat
     5161 ggcagcgtgc agctggctga tcactaccag caaaacactc caatcggtga tggtcctgtt
     5221 ctgctgccag acaatcacta tctgagcacg caaagcgttc tgtctaaaga tccgaacgag
     5281 aaacgcgatc atatggttct gctggagttc gtaaccgcag cgggcatcac gcatggtatg
     5341 gatgaactgt acaaatgacc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     5401 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac
     5461 cttcgggtgg gcctttctgc gtttataggt ctcagctgag catgatacgg aaatctgctc
     5521 gtcagtggtg ctcacactga cgaatcatgt acagatcata ccgatgactg cctggcgact
     5581 cacaactaag caagacagcc ggaaccagcg ccggcgaaca ccactgcata tatggcatat
     5641 cacaacagtc caactagtgc actgcagtac agtttagctt gcctcgtccc cgccgggtca
     5701 cccggccagc gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc
     5761 aggggcatga tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt
     5821 tgcatccata cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctcca
     5881 gacctgcgag cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc
     5941 cctgtagaga aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc
     6001 ttttaaaatc ttgctaggat acagttctca catcacatcc gaacataaac aaaaatgggt
     6061 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     6121 gtgtccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     6181 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
     6241 tatgtttatc ggcactttgc atcggccgcg ctcccg
//
