LOCUS       sequence-110439-        5516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector to create a cassette for homologous recombination in S. 
            cerevisiae; adds a C-terminal tDimer2 Twin-Strep tag followed by 
            HIS5 selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5516)
  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 5516)
  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: pGS1623
FEATURES             Location/Qualifiers
     source          1..5516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             10..687
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MVASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGTLIYKVKFRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFL"
     CDS             730..1401
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTLIYKVKFRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQA
                     LKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHHL
                     FL"
     CDS             join(1405..1410,1411..1434,1435..1470,1471..1494)
                     /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      1525..1712
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1580..1604)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1787..2987
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        1787..2130
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2131..2784
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="MGRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHTNQ
                     KGEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQ
                     AMSNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLY
                     SFSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      2790..2987
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3091..3110)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3092..3110)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3197..3214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3368..3956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3448..3467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4127..4918,4919..4987))
                     /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     4750..4769
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4988..5092)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5160..5178
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5216..5238)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5338..5357
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5438..5456
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5438..5455
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 ttaattaaca tggtggcctc ctccgaggac gtcatcaaag agttcatgcg cttcaaggtg
       61 cgcatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
      121 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc
      181 gcctgggaca tcctgtcccc ccagttccag tacggctcca aggcgtacgt gaagcacccc
      241 gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
      301 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
      361 acgctgatct acaaggtgaa gttccgcggc accaacttcc cccccgacgg ccccgtaatg
      421 cagaagaaga ccatgggctg ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg
      481 ctgaagggcg agatccacca ggccctgaag ctgaaggacg gcggccacta cctggtggag
      541 ttcaagacca tctacatggc caagaagccc gtgcagctgc ccggctacta ctacgtggac
      601 accaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc
      661 tccgagggcc gccaccacct gttcctgggg catggcaccg gcagcaccgg cagcggcagc
      721 tccggcaccg cctcctccga ggacgtcatc aaagagttca tgcgcttcaa ggtgcgcatg
      781 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
      841 gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg
      901 gacatcctgt ccccccagtt ccagtacggc tccaaggcgt acgtgaagca ccccgccgac
      961 atccccgact acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     1021 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcacgctg
     1081 atctacaagg tgaagttccg cggcaccaac ttcccccccg acggccccgt aatgcagaag
     1141 aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag
     1201 ggcgagatcc accaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag
     1261 accatctaca tggccaagaa gcccgtgcag ctgcccggct actactacgt ggacaccaag
     1321 ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga gcgctccgag
     1381 ggccgccacc acctgttcct gggtagtgct tggagtcatc cacaattcga aaaaggtgga
     1441 ggttccggag gtggatcggg aggttctgcc tggtctcatc cacaatttga gaagtaaggt
     1501 accgcgcgcg ccacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa
     1561 taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga
     1621 aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagtat
     1681 gaggtcgctc ttattgacca cacctctacc ggcagatccg ctagggataa cagggtaata
     1741 tagatctgtt tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca
     1801 gaataccctc cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc
     1861 gtacatttag cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg
     1921 cacggcgcga agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc
     1981 cctcacagac gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta
     2041 ggatttgcca ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag
     2101 ttctcacatc acatccgaac ataaacaacc atgggtagga gggcttttgt agaaagaaat
     2161 acgaacgaaa cgaaaatcag cgttgccatc gctttggaca aagctccctt acctgaagag
     2221 tcgaatttta ttgatgaact tataacttcc aagcatacaa accaaaaggg agaacaagta
     2281 atccaagtag acacgggaat tggattcttg gatcacatgt atcatgcact ggctaaacat
     2341 gcaggctgga gcttacgact ttactcaaga ggtgatttaa tcatcgatga tcatcacact
     2401 gcagaagata ctgctattgc acttggtatt gcattcaagc aggctatgag taactttgcc
     2461 ggcgttaaaa gatttggaca tgcttattgt ccacttgacg aagctctttc tagaagcgta
     2521 gttgacttgt cgggacggcc ctatgctgtt atcgatttgg gattaaagcg tgaaaaggtt
     2581 ggggaattgt cctgtgaaat gatccctcac ttactatatt ccttttcggt agcagctgga
     2641 attactttgc atgttacctg cttatatggt agtaatgacc atcatcgtgc tgaaagcgct
     2701 tttaaatctc tggctgttgc catgcgcgcg gctactagtc ttactggaag ttctgaagtc
     2761 ccaagcacga agggagtgtt gtaaagagta ctgacaataa aaagattctt gttttcaaga
     2821 acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg
     2881 tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga
     2941 aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat
     3001 actaacgccg ccatccagtt taaacgagct cgaattcatc gatgatatca gatccactag
     3061 tggcctatgc ggccgcggat ctgccggtct ccctatagtg agtcgtatta atttcgataa
     3121 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3181 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3241 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3301 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3361 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3421 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3481 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3541 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg
     3601 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3661 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3721 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3781 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     3841 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3901 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3961 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4021 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4081 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4141 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4201 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4261 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4321 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4381 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4441 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4501 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4561 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4621 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4681 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4741 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4801 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4861 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4921 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4981 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5041 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5101 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5161 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     5221 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     5281 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     5341 catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct
     5401 acaattaata cataacctta tgtatcatac acatacgatt taggtgacac tatagaacgc
     5461 ggccgccagc tgaagcttcg tacgctgcag gtcgacggat cggtgacggt gctggt
//
