LOCUS       sequence-403474-        3956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For C-terminal tagging of a protein-of-interest with the ALFA tag 
            using S2/S3 primers in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3956)
  AUTHORS   Akhuli D, Dhar A, Viji AS, Bhojappa B, Palani S
  TITLE     ALIBY: ALFA Nanobody-Based Toolkit for Imaging and Biochemistry in 
            Yeast.
  JOURNAL   mSphere. 2022 Oct 26;7(5):e0033322. doi: 10.1128/msphere.00333-22. 
            Epub 2022 Oct 3.
  PUBMED    36190134
REFERENCE   2  (bases 1 to 3956)
  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: "mSphere."; 
            date: "2022-10-26"; volume: "7(5):e0033322. doi"; pages: " 
            10.1128/msphere.00333-22. Epub 2022 Oct 3"
COMMENT     Sequence Label: pABY-T1
FEATURES             Location/Qualifiers
     source          1..3956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..50)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(32..50)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(137..154)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(308..896)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(388..407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1067..1858,1859..1927))
                     /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     1690..1709
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1928..2032)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2100..2118
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2156..2178)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2278..2297
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2378..2396
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2378..2395
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2440..2478
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     gene            2527..3883
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2527..2870
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2871..3680
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2938..2957)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3686..3883
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 tggcctatgc ggccgcggat ctgccggtct ccctatagtg agtcgtatta atttcgataa
       61 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
      121 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
      181 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
      241 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
      301 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
      361 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
      421 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
      481 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
      541 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
      601 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
      661 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
      721 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
      781 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
      841 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
      901 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
      961 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1021 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1081 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     1141 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     1201 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     1261 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     1321 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     1381 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     1441 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     1501 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     1561 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     1621 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     1681 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     1741 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     1801 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     1861 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     1921 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     1981 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     2041 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     2101 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     2161 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     2221 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     2281 catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct
     2341 acaattaata cataacctta tgtatcatac acatacgatt taggtgacac tatagaacgc
     2401 ggccgccagc tgaagcttcg tacgctgcag gtcgacccat caagattaga agaggagctg
     2461 aggagaagat tgaccgagta aagatctgtt tagcttgcct cgtccccgcc gggtcacccg
     2521 gccagcgaca tggaggccca gaataccctc cttgacagtc ttgacgtgcg cagctcaggg
     2581 gcatgatgtg actgtcgccc gtacatttag cccatacatc cccatgtata atcatttgca
     2641 tccatacatt ttgatggccg cacggcgcga agcaaaaatt acggctcctc gctgcagacc
     2701 tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt gtccccacgc cgcgcccctg
     2761 tagagaaata taaaaggtta ggatttgcca ctgaggttct tctttcatat acttcctttt
     2821 aaaatcttgc taggatacag ttctcacatc acatccgaac ataaacaacc atgggtaagg
     2881 aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     2941 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     3001 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     3061 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     3121 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggcaaaacag
     3181 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     3241 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     3301 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     3361 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt
     3421 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     3481 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     3541 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     3601 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     3661 tgctcgatga gtttttctaa tcagtactga caataaaaag attcttgttt tcaagaactt
     3721 gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat
     3781 ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt
     3841 aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta
     3901 acgccgccat ccagtttaaa cgagctcgaa ttcatcgatg atatcagatc cactag
//
