LOCUS       sequence-403476-        3799 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 3799)
  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 3799)
  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-T3
FEATURES             Location/Qualifiers
     source          1..3799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(184..527)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             complement(576..614)
                     /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"
     promoter        complement(658..676)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(659..676)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(757..776)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     876..898
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(936..954)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1022..1126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1127..1195,1196..1987)
                     /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     complement(1345..1364)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2158..2746
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2647..2666
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2900..2917
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3004..3023
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3004..3022
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(3215..3402)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     3323..3347
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(join(3410..3799,1..183))
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
ORIGIN
        1 ggggaacacc ttggtcaggg gcgggtccac cggcacctcc cgcagggtga agccgtcccc
       61 ggtggcggtg acgcggaaga cggtgtcggt ggtgaaggac ccatccagtg cctcgatggc
      121 ctcggcgtcc cccgggacac tggtgcggta ccggtaagcc gtgtcgtcaa gagtggtacc
      181 catggttgtt tatgttcgga tgtgatgtga gaactgtatc ctagcaagat tttaaaagga
      241 agtatatgaa agaagaacct cagtggcaaa tcctaacctt ttatatttct ctacaggggc
      301 gcggcgtggg gacaattcaa cgcgtctgtg aggggagcgt ttccctgctc gcaggtctgc
      361 agcgaggagc cgtaattttt gcttcgcgcc gtgcggccat caaaatgtat ggatgcaaat
      421 gattatacat ggggatgtat gggctaaatg tacgggcgac agtcacatca tgcccctgag
      481 ctgcgcacgt caagactgtc aaggagggta ttctgggcct ccatgtcgct ggccgggtga
      541 cccggcgggg acgaggcaag ctaaacagat ctttactcgg tcaatcttct cctcagctcc
      601 tcttctaatc ttgatgggtc gacctgcagc gtacgaagct tcagctggcg gccgcgttct
      661 atagtgtcac ctaaatcgta tgtgtatgat acataaggtt atgtattaat tgtagccgcg
      721 ttctaacgac aatatgtcca tatggtgcac tctcagtaca atctgctctg atgccgcata
      781 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
      841 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
      901 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
      961 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     1021 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     1081 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     1141 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     1201 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     1261 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     1321 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     1381 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     1441 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     1501 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1561 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1621 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1681 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1741 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1801 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1861 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1921 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1981 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     2041 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     2101 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2161 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2221 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2281 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     2341 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2401 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2461 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2521 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2581 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2641 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2701 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2761 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2821 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2881 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     2941 caaaccgcct ctccccgcgc gttggccgat tcattaatgc aggttaacct ggcttatcga
     3001 aattaatacg actcactata gggagaccgg cagatccgcg gccgcatagg ccactagtgg
     3061 atctgatatc atcgatgaat tcgagctcga ttacaacagg tgttgtcctc tgaggacata
     3121 aaatacacac cgagattcat caactcattg ctggagttag catatctaca attgggtgaa
     3181 atggggagcg atttgcaggc atttgctcgg catgccggta gaggtgtggt caataagagc
     3241 gacctcatgc tatacctgag aaagcaacct gacctacagg aaagagttac tcaagaataa
     3301 gaattttcgt tttaaaacct aagagtcact ttaaaatttg tatacactta ttttttttat
     3361 aacttattta ataataaaaa tcataaatca taagaaattc gcctcgagat taggggcagg
     3421 gcatgctcat gtagagcgcc tgctcgccgt ccgaggcggt gccgtcgtac agggcggtgt
     3481 ccaggccgca gagggtgaac cccatccgcc ggtacgcgtg gatcgccggt gcgttgacgt
     3541 tggtgacctc cagccagagg tgcccggcgc cccgctcgcg ggcgaactcc gtcgcgagcc
     3601 ccatcaacgc gcgcccgacc ccgtgccccc ggtgctccgg ggcgacctcg atgtcctcga
     3661 cggtcagccg gcggttccag ccggagtacg agacgaccac gaagcccgcc aggtcgccgt
     3721 cgtccccgta cgcgacgaac gtccgggagt ccgggtcgcc gtcctccccg tcgtccgatt
     3781 cgtcgtccga ttcgtcgtc
//
