LOCUS Exported 5764 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION FX cloning Pichia pastoris expression vector with AOX1 promoter and N-terminal 10x His tag, GFP and 3C protease cleavage site. ACCESSION . VERSION . KEYWORDS pICXNHG3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5764) AUTHORS Geertsma ER, Dutzler R TITLE A versatile and efficient high-throughput cloning tool for structural biology. JOURNAL Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25. PUBMED 21410291 REFERENCE 2 (bases 1 to 5764) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5764 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 2..940 /gene="Pichia pastoris AOX1" /label=AOX1 promoter /note="inducible promoter, regulated by methanol" CDS 985..1014 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 1015..1725 /codon_start=1 /product="yeast-enhanced green fluorescent protein" /label=yeGFP /note="codons optimized for translation in S. cerevisiae and C. albicans" /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHGMDELYK" CDS 1732..1755 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS complement(1804..2109) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(1906..1925) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(2451..3110) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 3025..3044 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(3164..3194) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind complement(3219..3343) /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(3362..3382) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(3386..3406) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3446..3475 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 3491..3508 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 3588..3834 /gene="Pichia pastoris AOX1" /label=AOX1 terminator /note="transcription terminator for AOX1" promoter 3849..4260 /gene="S. cerevisiae TEF1" /label=TEF1 promoter /note="promoter for EF-1-alpha" promoter 4268..4315 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 4334..4708 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" terminator 4774..5021 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(4790..4808) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" rep_origin complement(5096..5684) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5176..5195) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 901 caacttgaga agatcaaaaa acaactaatt attcgaaacg aggaattcac gtggcccagc 961 cggccgtctc ggatcggtac catgcaccat catcatcacc atcatcatca tcattctaaa 1021 ggagaagaat tattcactgg tgttgtccca attttggttg aattagatgg tgatgttaat 1081 ggtcacaaat tttctgtctc cggtgaaggt gaaggtgatg ctacttacgg taaattgacc 1141 ttaaaattta tttgtactac tggtaaattg ccagttccat ggccaacctt agtcactact 1201 ttcggttatg gtgttcaatg ttttgcgaga tacccagatc atatgaaaca acatgacttt 1261 ttcaagtctg ccatgccaga aggttatgtt caagaaagaa ctattttttt caaagatgac 1321 ggtaactaca agaccagagc tgaagtcaag tttgaaggtg ataccttagt taatagaatc 1381 gaattaaaag gtattgattt taaagaagat ggtaacattt taggtcacaa attggaatac 1441 aactataact ctcacaatgt ttacatcatg gctgacaaac aaaagaatgg tatcaaagtt 1501 aacttcaaaa ttagacacaa cattgaagat ggttctgttc aattagctga ccattatcaa 1561 caaaatactc caattggtga tggtccagtc ttgttaccag acaaccatta cttatccact 1621 caatctgcct tatccaaaga tccaaacgaa aagagagacc acatggtctt gttagaattt 1681 gttactgctg ctggtattac ccatggtatg gatgaattgt acaaaactag tttagaagtt 1741 ttgtttcaag gtccaagtag aagagcgagc tgcagactgg ctgtgtataa gggagcctga 1801 catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct 1861 gagatcagcc acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat 1921 catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc 1981 tgacagcaga cgtgcactgg ccagggggat caccatccgt cgcccgggcg tgtcaataat 2041 atcactctgt acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt 2101 aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc 2161 gggcgacctc agccatccct tcctgatttt ccgctttcca gcgttcggca cgcagacgac 2221 gggcttcatt ctgcatggtt gtgcttacca gaccggagat attgacatca tatatgcctt 2281 gagcaactga tagctgtcgc tgtcaactgt cactgtaata cgctgcttca tagcatacct 2341 ctttttgaca tacttcgggt atacatatca gtatatattc ttataccgca aaaatcagcg 2401 cgcaaatacg catactgtta tctggctttt agtaagccgg atccacgcgt ttacgccccg 2461 ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca 2521 tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta 2581 taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa 2641 tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt ctcaataaac 2701 cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt 2761 agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt ttcagtttgc 2821 tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc 2881 attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc 2941 ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga 3001 acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga 3061 tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc 3121 ttagctcctg aaaatctcga cggatcctaa ctcaaaatcc acacattata cgagccggaa 3181 gcataaagtg taaagcctgg ggtgcctaat gcggccgcca tagtgactgg atatgttgtg 3241 ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta 3301 tatcatttta cgtttctcgt tcagcttttt tgtacaaact tgtgatatcg aattcgctag 3361 ctacctttct cttctttttt ggatctacct ttctcttctt ttttggggat ccactagttc 3421 agctcttctg cataataact atctagaaca aaaactcatc tcagaagagg atctgaatag 3481 cgccgtcgac catcatcatc atcatcattg agtttgtagc cttagacatg actgttcctc 3541 agttcaagtt gggcacttac gagaagaccg gtcttgctag attctaatca agaggatgtc 3601 agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta tttgtaacct 3661 atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg ctcctgatca 3721 gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca ttcgagtttg 3781 atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt gagaccttcg 3841 tttgtgcgga tcccccacac accatagctt caaaatgttt ctactccttt tttactcttc 3901 cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag cacagcatac 3961 taaattttcc ctctttcttc ctctagggtg tcgttaatta cccgtactaa aggtttggaa 4021 aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata aaaattttta 4081 tcacgtttct ttttcttgaa attttttttt ttagtttttt tctctttcag tgacctccat 4141 tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt 4201 ctattacaac tttttttact tcttgttcat tagaaagaaa gcatagcaat ctaatctaag 4261 gggcggtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt 4321 gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt 4381 cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga 4441 cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca 4501 ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc 4561 cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga 4621 gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt 4681 gcacttcgtg gccgaggagc aggactgaca cgtccgacgg cggcccacgg gtcccaggcc 4741 tcggagatcc gtcccccttt tcctttgtcg atatcatgta attagttatg tcacgcttac 4801 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag 4861 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc 4921 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct 4981 tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagctggag accaacatgt 5041 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 5101 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 5161 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 5221 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 5281 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 5341 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 5401 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 5461 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 5521 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 5581 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 5641 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 5701 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 5761 gatc //