LOCUS       sequence-82002-e        4947 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For C-terminal tagging of ORFs with a (3×FLAG)-TEV-ProtA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4947)
  TITLE     various
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4947)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pFA6a-hphMX-(3×FLAG)-TEV-ProtA
FEATURES             Location/Qualifiers
     source          1..4947
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(79..870,871..939))
                     /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     702..721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(940..1044)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1112..1130
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1168..1190)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1290..1309
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1390..1408
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1390..1407
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1487..1558
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             1586..1606
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1646..1819
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             1820..1993
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     terminator      2047..2234
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2102..2126)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2309..3884
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        2309..2652
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2653..3681
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2653..3681
                     /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"
     terminator      3687..3884
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3990..4009)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3991..4009)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4096..4113)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4267..4855)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4347..4366)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
       61 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
      121 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
      181 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
      241 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
      301 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
      361 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
      421 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
      481 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
      541 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
      601 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
      661 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
      721 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
      781 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
      841 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
      901 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
      961 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     1021 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     1081 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     1141 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     1201 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     1261 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     1321 ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat
     1381 acacatacga tttaggtgac actatagaac gcggccgcca gctgaagctt cgtacgctgc
     1441 aggtcgacgg atccccgggt taattaatat gtctttaatc aatatggact acaaggacga
     1501 cgatgacaag gattacaaag atgacgacga taaggactat aaggacgatg atgataaaga
     1561 ttatgatatt ccaactactg ctagcgagaa tttgtatttt cagggtgagc tcaaaaccgc
     1621 ggctcttgcg caacacgatg aagccgtgga caacaaattc aacaaagaac aacaaaacgc
     1681 gttctatgag atcttacatt tacctaactt aaacgaagaa caacgaaacg ccttcatcca
     1741 aagtttaaaa gatgacccaa gccaaagcgc taacctttta gcagaagcta aaaagctaaa
     1801 tgatgctcag gcgccgaaag tagacaacaa attcaacaaa gaacaacaaa acgcgttcta
     1861 tgagatctta catttaccta acttaaacga agaacaacga aacgccttca tccaaagttt
     1921 aaaagatgac ccaagccaaa gcgctaacct tttagcagaa gctaaaaagc taaatggtgc
     1981 tcaggcgccg aaagtagacg cgaattccgc ggggaagtca acctgaggcg cgccacttct
     2041 aaataagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata
     2101 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt
     2161 aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc tcttattgac
     2221 cacacctcta ccggcagatc cgctagggat aacagggtaa tatagatctg tttagcttgc
     2281 cttgtccccg ccgggtcacc cggccagcga catggaggcc cagaataccc tccttgacag
     2341 tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc ccgtacattt agcccataca
     2401 tccccatgta taatcatttg catccataca ttttgatggc cgcacggcgc gaagcaaaaa
     2461 ttacggctcc tcgctgcaga cctgcgagca gggaaacgct cccctcacag acgcgttgaa
     2521 ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt taggatttgc cactgaggtt
     2581 cttctttcat atacttcctt ttaaaatctt gctaggatac agttctcaca tcacatccga
     2641 acataaacaa ccatgggtaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     2701 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     2761 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     2821 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     2881 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     2941 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     3001 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     3061 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     3121 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     3181 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     3241 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     3301 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     3361 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     3421 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     3481 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     3541 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     3601 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     3661 cgtccgaggg caaaggaata atcagtactg acaataaaaa gattcttgtt ttcaagaact
     3721 tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga
     3781 tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag
     3841 taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact
     3901 aacgccgcca tccagtgttt aaacgagctc gaattcatcg atgatatcag atccactagt
     3961 ggcctatgcg gccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag
     4021 ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4081 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4141 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4201 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4261 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4321 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4381 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4441 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4501 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4561 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4621 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4681 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     4741 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4801 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4861 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4921 tttggtcatg agattatcaa aaaggat
//
