LOCUS Exported 4492 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-natMX-(3×FLAG)-TEV-ProtA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4492) TITLE various JOURNAL Unpublished REFERENCE 2 (bases 1 to 4492) AUTHORS .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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4492 /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..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 //