LOCUS Exported 4947 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For C-terminal tagging of ORFs with a (3×FLAG)-TEV-ProtA. ACCESSION . VERSION . KEYWORDS pFA6a-hphMX-(3×FLAG)-TEV-ProtA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4947) TITLE various JOURNAL Unpublished REFERENCE 2 (bases 1 to 4947) AUTHORS .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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4947 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(79..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 1823..1993 /codon_start=1 /product="IgG-binding unit of Staphylococcus aureus protein A" /label=ProtA /translation="DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA EAKKLNGAQAPK" 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 /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 //