LOCUS       sequence-106478-        5000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector to create a cassette for homologous recombination in S. 
            cerevisiae; adds a C-terminal eGFP Twin-Strep tag followed by URA3 
            selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5000)
  AUTHORS   Rai J, Pemmasani JK, Voronovsky A, Jensen IS, Manavalan A, Nyengaard
            JR, Golas MM, Sander B
  TITLE     Strep-tag II and Twin-Strep Based Cassettes for Protein Tagging by 
            Homologous Recombination and Characterization of Endogenous 
            Macromolecular Assemblies in Saccharomyces cerevisiae.
  JOURNAL   Mol Biotechnol. 2014 Jun 27.
  PUBMED    24969434
REFERENCE   2  (bases 1 to 5000)
  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: "Mol 
            Biotechnol. 2014 Jun 27."
COMMENT     Sequence Label: pGS1222
FEATURES             Location/Qualifiers
     source          1..5000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(61..80)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(62..80)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(167..184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(338..926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(418..437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1097..1888,1889..1957))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1720..1739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1958..2062)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2130..2148
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2186..2208)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2308..2327
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2408..2426
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2408..2425
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2496..3209
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2496..3209
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(3213..3218,3219..3242,3243..3278,3279..3302)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     terminator      3333..3520
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3388..3412)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        3595..3938
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      4757..4954
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 cgaattcatc gatgatatca gatccactag tggcctatgc ggccgcggat ctgccggtct
       61 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
      121 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
      181 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
      241 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
      301 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
      361 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
      421 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
      481 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac
      541 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
      601 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
      661 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
      721 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
      781 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
      841 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
      901 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
      961 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     1021 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     1081 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     1141 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     1201 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     1261 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     1321 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     1381 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     1441 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     1501 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     1561 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     1621 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     1681 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     1741 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     1801 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     1861 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     1921 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     1981 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2041 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     2101 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     2161 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2221 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2281 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     2341 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
     2401 acatacgatt taggtgacac tatagaacgc ggccgccagc cgaagcttcg tacgctgcag
     2461 gtcgacggat cggtgacggt gctggtttaa ttaacatgtc taaaggtgaa gaattattca
     2521 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
     2581 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     2641 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaact tatggtgttc
     2701 aatgtttttc tagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     2761 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     2821 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     2881 attttaaaga agatggtaac attttaggtc acaaattgga atacaactat aactctcaca
     2941 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     3001 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     3061 gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct gccttatcca
     3121 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     3181 ttacccatgg tatggatgaa ttgtacaaag gtagtgcttg gagtcatcca caattcgaaa
     3241 aaggtggagg ttccggaggt ggatcgggag gttctgcctg gtctcatcca caatttgaga
     3301 agtaaggtac cgcgcgcgcc acttctaaat aagcgaattt cttatgattt atgattttta
     3361 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     3421 taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg
     3481 tatagtatga ggtcgctctt attgaccaca cctctaccgg cagatccgct agggataaca
     3541 gggtaatata gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg
     3601 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac
     3661 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt
     3721 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcggacctg cgagcaggga
     3781 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata
     3841 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta
     3901 ggatacagtt ctcacatcac atccgaacat aaacaaccat gacagtcaac actaagacct
     3961 atagtgagag agcagaaact catgcctcac cagtagcaca acgattattt cgattaatgg
     4021 aactgaagaa aaccaattta tgtgcatcaa ttgatgttga taccantaag gaattccttg
     4081 aattaattga taaattgggt cnttatgtat gcttaatcaa gactcatatt gatataatca
     4141 atgatttttc ctatgaatcc actattgaac cattattaga actttcacgt aaacatcaat
     4201 ttatgatttt tgaagataga aaatttgctg atattggtaa taccgtgaag aaacaatata
     4261 ttggtggagt ttataaaatt agtagttggg cagatattac taatgctcat ggtgtcactg
     4321 ggaatggagt agttgaagga ttaaaacagg gagctaaaga aaccaccacc aaccaagagc
     4381 caagagggtt attgatgtta gctgaattat catcagtggg atcattagca tatggagaat
     4441 attctcaaaa aactgttgaa attgctaaat ccgataagga atttgttatt ggatttattg
     4501 cccaacgtga tatgggtgga caagaagaag gatttgattg gcttattatg acacctggag
     4561 ttggattaga tgataaaggt gatggattag gacaacaata tagaactgtt gatgaagttg
     4621 ttagcactgg aactgatatt atcattgttg gtagaggatt gtttggtaaa ggaagagatc
     4681 cagatattga aggtaaaagg tatagagatg ctggttggaa tgcttatttg aaaaagactg
     4741 gccaattata atctgtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt
     4801 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt
     4861 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg
     4921 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca
     4981 tccagtgtcg aaaacgagct
//
