LOCUS       sequence-82003-e        4492 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 4492)
  TITLE     various
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4492)
  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            "Unpublished"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-natMX-(3×FLAG)-TEV-ProtA
FEATURES             Location/Qualifiers
     source          1..4492
                     /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..3431
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2309..2652
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2653..3228
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVISYSAWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFAGERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGERQALYMSMPCP"
     terminator      3234..3431
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3535..3554)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3536..3554)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3641..3658)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3812..4400)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3892..3911)
                     /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 ctcgtccccg 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 ccatgggtac cactcttgac gacacggctt accggtaccg caccagtgtc
     2701 ccgggggacg ccgaggccat cgaggcactg gatgggtcct tcaccaccga caccgtcttc
     2761 cgcgtcaccg ccaccgggga cggcttcacc ctgcgggagg tgccggtgga cccgcccctg
     2821 accaaggtgt tccccgacga cgaatcggac gacgaatcgg acgacgggga ggacggcgac
     2881 ccggactccc ggacgttcgt cgcgtacggg gacgacggcg acctggcggg cttcgtggtc
     2941 atctcgtact cggcgtggaa ccgccggctg accgtcgagg acatcgaggt cgccccggag
     3001 caccgggggc acggggtcgg gcgcgcgttg atggggctcg cgacggagtt cgccggcgag
     3061 cggggcgccg ggcacctctg gctggaggtc accaacgtca acgcaccggc gatccacgcg
     3121 taccggcgga tggggttcac cctctgcggc ctggacaccg ccctgtacga cggcaccgcc
     3181 tcggacggcg agcggcaggc gctctacatg agcatgccct gcccctaatc agtactgaca
     3241 ataaaaagat tcttgttttc aagaacttgt catttgtata gtttttttat attgtagttg
     3301 ttctatttta atcaaatgtt agcgtgattt atattttttt tcgcctcgac atcatctgcc
     3361 cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg
     3421 tcgctatact gctgtcgatt cgatactaac gccgccatcc agtttaaacg agctcgaatt
     3481 catcgatgat atcagatcca ctagtggcct atgcggccgc ggatctgccg gtctccctat
     3541 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     3601 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3661 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3721 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3781 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3841 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3901 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3961 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
     4021 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4081 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4141 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4201 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4261 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4321 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4381 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4441 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg at
//
