LOCUS       sequence-403478-        3800 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 3800)
  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 3800)
  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-T4
FEATURES             Location/Qualifiers
     source          1..3800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(82..99)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(253..841)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(333..352)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1012..1803,1804..1872))
                     /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     1635..1654
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1873..1977)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2045..2063
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2101..2123)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2223..2242
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2323..2341
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2323..2340
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2385..2423
                     /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            2472..3672
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        2472..2815
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2816..3469
                     /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="MGRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHANQ
                     KGEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQ
                     AMGNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLY
                     SFSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      3475..3672
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3776..3795)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3777..3795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
       61 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
      121 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
      181 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
      241 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
      301 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
      361 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
      421 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
      481 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
      541 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
      601 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
      661 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
      721 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
      781 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
      841 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
      901 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
      961 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1021 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     1081 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     1141 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     1201 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     1261 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     1321 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     1381 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     1441 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     1501 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     1561 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     1621 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     1681 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     1741 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     1801 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     1861 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     1921 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     1981 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     2041 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     2101 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     2161 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     2221 tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg
     2281 cggctacaat taatacataa ccttatgtat catacacata cgatttaggt gacactatag
     2341 aacgcggccg ccagctgaag cttcgtacgc tgcaggtcga cccatcaaga ttagaagagg
     2401 agctgaggag aagattgacc gagtaaagat ctgtttagct tgcctcgtcc ccgccgggtc
     2461 acccggccag cgacatggag gcccagaata ccctccttga cagtcttgac gtgcgcagct
     2521 caggggcatg atgtgactgt cgcccgtaca tttagcccat acatccccat gtataatcat
     2581 ttgcatccat acattttgat ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc
     2641 agacctgcga gcagggaaac gctcccctca cagacgcgtt gaattgtccc cacgccgcgc
     2701 ccctgtagag aaatataaaa ggttaggatt tgccactgag gttcttcttt catatacttc
     2761 cttttaaaat cttgctagga tacagttctc acatcacatc cgaacataaa caaccatggg
     2821 taggagggct tttgtagaaa gaaatacgaa cgaaacgaaa atcagcgttg ccatcgcttt
     2881 ggacaaagct cccttacctg aagagtcgaa ttttattgat gaacttataa cttccaagca
     2941 tgcaaaccaa aagggagaac aagtaatcca agtagacacg ggaattggat tcctggatca
     3001 catgtatcat gcactggcta aacatgcagg ctggagctta cgactttact caagaggtga
     3061 tttaatcatc gatgatcatc acactgcaga agatactgct attgcacttg gtattgcatt
     3121 caagcaggct atgggtaact ttgccggcgt taaaagattt ggacatgctt attgtccact
     3181 tgacgaagcc ctttctagaa gcgtagttga cttgtcggga cggccctatg ctgttatcga
     3241 tttgggatta aagcgtgaaa aggttgggga attgtcctgt gaaatgatcc ctcacttact
     3301 atattccttt tcggtagcag ctggaattac tttgcatgtt acctgcttat atggtagtaa
     3361 tgaccatcat cgtgctgaaa gcgcttttaa atctctggct gttgccatgc gcgcggctac
     3421 tagtcttact ggaagttctg aagtcccaag cacgaaggga gtgttgtaaa gagtactgac
     3481 aataaaaaga ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt
     3541 gttctatttt aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc
     3601 ccagatgcga agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg
     3661 gtcgctatac tgctgtcgat tcgatactaa cgccgccatc cagtttaaac gagctcgaat
     3721 tcatcgatga tatcagatcc actagtggcc tatgcggccg cggatctgcc ggtctcccta
     3781 tagtgagtcg tattaatttc
//
