LOCUS       sequence-403475-        4185 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 4185)
  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 4185)
  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-T2
FEATURES             Location/Qualifiers
     source          1..4185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(28..66)
                     /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(110..128)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(111..128)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(209..228)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     328..350
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(388..406)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        474..578
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(579..647,648..1439)
                     /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(797..816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1610..2198
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2099..2118
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2352..2369
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2456..2475
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2456..2474
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2537..2784)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     2750..2768
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(2792..3820)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(2792..3820)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(3821..4164)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
ORIGIN
        1 ggacgaggca agctaaacag atctttactc ggtcaatctt ctcctcagct cctcttctaa
       61 tcttgatggg tcgacctgca gcgtacgaag cttcagctgg cggccgcgtt ctatagtgtc
      121 acctaaatcg tatgtgtatg atacataagg ttatgtatta attgtagccg cgttctaacg
      181 acaatatgtc catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
      241 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
      301 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
      361 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
      421 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
      481 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      541 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      601 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      661 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      721 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
      781 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
      841 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
      901 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
      961 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1021 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1081 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     1141 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1201 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1261 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     1321 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1381 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1441 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1501 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1561 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1621 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1681 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1741 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1801 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1861 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1921 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1981 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2041 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2101 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2161 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2221 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2281 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2341 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     2401 ctctccccgc gcgttggccg attcattaat gcaggttaac ctggcttatc gaaattaata
     2461 cgactcacta tagggagacc ggcagatccg cggccgcata ggccactagt ggatctgata
     2521 tcatcgatga attcgagctc gttaaagcct tcgagcgtcc caaaaccttc tcaagcaagg
     2581 ttttcagtat aatgttacat gcgtacacgc gtctgtacag aaaaaaaaga aaaatttgaa
     2641 atataaataa cgttcttaat actaacataa ctataaaaaa ataaataggg acctagactt
     2701 caggttgtct aactccttcc ttttcggtta gagcggatgt ggggggaggg cgtgaatgta
     2761 agcgtgacat aactaattac atgactcgag attattcctt tgccctcgga cgagtgctgg
     2821 ggcgtcggtt tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc
     2881 ttctgcgggc gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc
     2941 atcgaccctg cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt
     3001 ggtcaagacc aatgcggagc atatacgccc ggagccgcgg cgatcctgca agctccggat
     3061 gcctccgctc gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga
     3121 agatgttggc gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg
     3181 ctgttatgcg gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt
     3241 gccggacttc ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg
     3301 acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg
     3361 cgcatatgaa atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga
     3421 acccgctcgt ctggctaaga tcggccgcag cgatcgcatc catggcctcc gcgaccggct
     3481 gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc
     3541 gggagatgca ataggtcagg ctctcgctga attccccaat gtcaagcact tccggaatcg
     3601 ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc
     3661 agctatttac ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt
     3721 cttcgccctc cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact
     3781 tctcgacaga cgtcgcggtg agttcaggct ttttacccat ggttgtttat gttcggatgt
     3841 gatgtgagaa ctgtatccta gcaagatttt aaaaggaagt atatgaaaga agaacctcag
     3901 tggcaaatcc taacctttta tatttctcta caggggcgcg gcgtggggac aattcaacgc
     3961 gtctgtgagg ggagcgtttc cctgctcgca ggtccgcagc gaggagccgt aatttttgct
     4021 tcgcgccgtg cggccatcaa aatgtatgga tgcaaatgat tatacatggg gatgtatggg
     4081 ctaaatgtac gggcgacagt cacatcatgc ccctgagctg cgcacgtcaa gactgtcaag
     4141 gagggtattc tgggcctcca tgtcgctggc cgggtgaccc ggcgg
//
