LOCUS pHW564_Pmyo_2_cG 4666 bp ds-DNA circular 21-JAN-2018 DEFINITION . ACCESSION VERSION SOURCE . ORGANISM . COMMENT pHW564 Pmyo-2::NLS::cGAL(DBD)::gp41-1-N-intein(C1A)::let-858 3'UTR COMMENT (C1A mutation block the N-terminal cleavage, (C-terminal cleavge is OK, but lower rate, see the reference of the unprecedent fast intein, 2012) COMMENT The clone of pHW564(2.2) and L2.1 clones were confirmed by sequencing (largen 147517, 20171116 and L147682 at 20171117,) with oHW10f and oH233r COMMENT Sequence with oHW464f with both colonies to confirm the C termina of intein, at 20171218, Laragen 149112. And the resutls show that both L2.1 and L2.2 clones are confirmed to be correct at the C terminal of Int-N are correct COMMENT Save pHW564(2.2) COMMENT Vector: pAH35 (AvrII /KpnI) COMMENT Insert: 350bp PCR product gp41-1 N intein (C1A) mutation from pAH35 (gp41-1 N intein WT), size ~350bp with oHW464f + oHW465r (then digest with AvrII and KpnI) COMMENT T4 ligation COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers primer_bind 4521..4541 /label=M13-rev /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_signal 1103..1126 /label=NLS /ApEinfo_fwdcolor=#ff80ff /ApEinfo_revcolor=#66ffcc /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 rep_origin 3471..4153 /label=ColE1 origin /ApEinfo_fwdcolor=gray50 /ApEinfo_revcolor=gray50 /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_binding 4493..4515 /label=LacO /ApEinfo_fwdcolor=#6495ed /ApEinfo_revcolor=#6495ed /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 37..64 /label=p.myo2F2 primer /ApEinfo_fwdcolor=#ff8040 /ApEinfo_revcolor=#ff8040 /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 CDS 2714..3373 /label=AmpR /ApEinfo_fwdcolor=yellow /ApEinfo_revcolor=yellow /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 976..998 /label=p.myo2R2 primer /ApEinfo_fwdcolor=#004080 /ApEinfo_revcolor=#004080 /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 1616..1618 /label=gp41 N-intein (C1A) variant C elegan codon optimzat /ApEinfo_fwdcolor=#008040 /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 3'UTR 1919..2351 /label=let-858 3UTR /ApEinfo_fwdcolor=#bfddbb /ApEinfo_revcolor=#bfddbb /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 1616..1618 /label=gp41-1 (C1A)mutantion, C elegan codon optimzed. /ApEinfo_fwdcolor=#008000 /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 37..998 /label=pMyo-2 /ApEinfo_fwdcolor=#8000ff /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 1616..1618 /label=C1A (TGC to gct) /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 promoter join(4606..4666,1..2) /label=decoy ORF /ApEinfo_fwdcolor=#999999 /ApEinfo_revcolor=#999999 /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 intron 4556..4605 /label=decoy intron /ApEinfo_fwdcolor=#b3b3b3 /ApEinfo_revcolor=#b3b3b3 /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 CDS 1133..1573 /label=SK GAL4 /ApEinfo_fwdcolor=#ff8040 /ApEinfo_revcolor=#00ff80 /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 CDS 1619..1879 /label=gp41-1 N-intein C1A /ApEinfo_fwdcolor=#00ffff /ApEinfo_revcolor=#00ff80 /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 ORIGIN 1 AAGCTTgcat gcgcggccgc actgactgGG CCGGCCgtag tatcctttgc tttaaatgtc 61 cataaaacta attttataat caataaaaca acgtttgtaa atcaactgag tttacaagta 121 gagacattga gggatacttt cactatgcta aagtgataat cgaccaaata ataacttcac 181 tttggtattt attcctgtct tataaatgtt atgtatgaat taaattcata tgcatatggc 241 tcactctgac aatttttaat aatcttccag atcaatattg actaccgatg cgggtggtct 301 tttgctttga attctgctga actttacacc ccgaacagca atgtgtgctt cagcctaaaa 361 aaaagtaagt gtgttaatca gtgcccccga ttcttcattt tttgcccctc tctcccgttt 421 cgtcggcaaa agaagagaaa ataaagataa gtctcaagat aggttggtaa tcgctaaagt 481 ggttgtgtgg ataagagtag caaaatggca ggaagagcac tttgcgcgca cacactgtac 541 tcattgttct ggataaaatt ctctcgttgt ttgccgtcgg atgtctgcct ctctgcattg 601 agccggcttc ttcactatct ttagttaacc taaaatgccg tttcttttct cgtatcccca 661 ctatcccgtt gaggttctct gctctcttcg ctccctaccg ccagcgagca actatccgtg 721 ggggcgcctt gctcggaaga tgggggggaa gaaagaagat ttttgctatt tgcacttgag 781 aaagagactt ttcctgcgtc gatggttaga gaacagtgtg cagacacttt tcagctacct 841 agaattacaa ttggatatcc ccgcctccca atccacccac ccagggaaaa agaagggctc 901 gccgaaaatc aaagttatct ccaggctcgc gcatcccacc gagcggttga cttctctcca 961 ccacttttca ttttaaccct cgatcgtcag acacagaaGG CGCGCCtcta gaggatcccc 1021 gggattggcc aaaggaccca aaggtatgtt tcgaatgata ctaacataac atagaacatt 1081 ttcaggagga cccttgGCTA GGatgggccc taaaaagaag cgtaaaGCTA GCATGAAGCT 1141 GTTGTCTTCA ATGGAACAAG CTTGCGACAT ATGCCGGTTA AAAAAACTCA GGTGCTCGAA 1201 GGAAAAGCCC AAGTGTGCCA AGTGTCTGAA GAACAACTCG GAGTGCCGTT ATTCTCCCAA 1261 GGCTATGAGG TCCCCACTGA CCAGGGCGCA TCTGACAGAG GTAGAATCAA GGTTGGAAAA 1321 ATTGGAAGAT TTGTTTCTGC TCATGTTTCC TCGGGAAAAC CTGGACAGCA TTTTGAACAT 1381 GGACTCTTTG GACGAAGTGA AAGTAATGTT GAAACAATTA TATATGCAGG ACTATGGTGA 1441 CACGGATGCT GCTATGGATA GCTTGACCCC TATGGAAAAT GGCATGTCTG AAGGGCCGAG 1501 AAAACACATG AAGACTGCGA CATCATCGCC AAAAGTGGAT GAAGATGGAA GCCAAAGTCA 1561 GTTGAGTGTA TCGccggaat tcccgCTGCA Gtctggtggc ggaggggctC CTAGGgctCT 1621 CGACCTCAAG ACCCAAGTCC AAACCCCACA AGGAATGAAG GAGATCTCCA ACATCCAAGT 1681 CGGAGACCTC GTCCTCTCCA ACACCGGATA CAACGAGGTC CTCAACGTCT TCCCAAAGTC 1741 CAAGAAGAAG TCCTACAAGA TCACCCTCGA GGACGGAAAG GAGATCATCT GCTCCGAGGA 1801 GCACCTCTTC CCAACCCAAA CCGGAGAGAT GAACATCTCC GGAGGACTCA AGGAGGGAAT 1861 GTGCCTCTAC GTCAAGGAGT AAGGTACCGA ATTCGCTAGC CGGCCATACA AGTAATCCGG 1921 ATGATCGACG CCaACGTCGT TGAATTTTCA AATTTTAAAT ACTGAATATT TGTTTTTTTT 1981 CCTATTATTT ATTTATTCTC TTTGTGTTTT TTTTCTTGCT TTCTAAAAAA TTAATTCAAT 2041 CCAAATCTAA acattttttt ttctctttcc gtctcccaat tcgtattccg ctcctctcat 2101 ctgaacacaa tgtgcaagtt tatttatctt ctcgctttca tttcattagg acgtgggggg 2161 aattggtgga agggggaaac acacaaaagg atgatggaaa tgaaataagg acacacaata 2221 tgcaacaaca ttcaattcag aaatatggag gaaggtttaa aagaaaacat aaaaatatat 2281 agaggaggaa ggaaaactag taaaaaataa gcaaagaaat taggcgaacg atgAGAATTG 2341 TCCTCGCTTG GGCCCAAAAG GCCTTTTTTT TTTGGCGCCG ACGTCAGGTG GCACTTTTCG 2401 GGGAAATGTG CGCGGAACCC CTATTTGTTT ATTTTTCTAA ATACATTCAA ATATGTATCC 2461 GCTCATGAGA CAATAACCCT GATAAATGCT TCAATAATAT TGAAAAAGGA AGAGTATGAG 2521 TATTCAACAT TTCCGTGTCG CCCTTATTCC CTTTTTTGCG GCATTTTGCC TTCCTGTTTT 2581 TGCTCACCCA GAAACGCTGG TGAAAGTAAA AGATGCTGAA GATCAGTTGG GTGCACGAGT 2641 GGGTTACATC GAACTGGATC TCAACAGCGG TAAGATCCTT GAGAGTTTTC GCCCCGAAGA 2701 ACGTTTTCCA ATGATGAGCA CTTTTAAAGT TCTGCTATGT GGCGCGGTAT TATCCCGTAT 2761 TGACGCCGGG CAAGAGCAAC TCGGTCGCCG CATACACTAT TCTCAGAATG ACTTGGTTGA 2821 GTACTCACCA GTCACAGAAA AGCATCTTAC GGATGGCATG ACAGTAAGAG AATTATGCAG 2881 TGCTGCCATA ACCATGAGTG ATAACACTGC GGCCAACTTA CTTCTGACAA CGATCGGAGG 2941 ACCGAAGGAG CTAACCGCTT TTTTGCACAA CATGGGGGAT CATGTAACTC GCCTTGATCG 3001 TTGGGAACCG GAGCTGAATG AAGCCATACC AAACGACGAG CGTGACACCA CGATGCCTGT 3061 AGCAATGGCA ACAACGTTGC GCAAACTATT AACTGGCGAA CTACTTACTC TAGCTTCCCG 3121 GCAACAATTA ATAGACTGGA TGGAGGCGGA TAAAGTTGCA GGACCACTTC TGCGCTCGGC 3181 CCTTCCGGCT GGCTGGTTTA TTGCTGATAA ATCTGGAGCC GGTGAGCGTG GGTCTCGCGG 3241 TATCATTGCA GCACTGGGGC CAGATGGTAA GCCCTCCCGT ATCGTAGTTA TCTACACGAC 3301 GGGGAGTCAG GCAACTATGG ATGAACGAAA TAGACAGATC GCTGAGATAG GTGCCTCACT 3361 GATTAAGCAT TGGTAACTGT CAGACCAAGT TTACTCATAT ATACTTTAGA TTGATTTAAA 3421 ACTTCATTTT TAATTTAAAA GGATCTAGGT GAAGATCCTT TTTGATAATC TCATGACCAA 3481 AATCCCTTAA CGTGAGTTTT CGTTCCACTG AGCGTCAGAC CCCGTAGAAA AGATCAAAGG 3541 ATCTTCTTGA GATCCTTTTT TTCTGCGCGT AATCTGCTGC TTGCAAACAA AAAAACCACC 3601 GCTACCAGCG GTGGTTTGTT TGCCGGATCA AGAGCTACCA ACTCTTTTTC CGAAGGTAAC 3661 TGGCTTCAGC AGAGCGCAGA TACCAAATAC TGTCCTTCTA GTGTAGCCGT AGTTAGGCCA 3721 CCACTTCAAG AACTCTGTAG CACCGCCTAC ATACCTCGCT CTGCTAATCC TGTTACCAGT 3781 GGCTGCTGCC AGTGGCGATA AGTCGTGTCT TACCGGGTTG GACTCAAGAC GATAGTTACC 3841 GGATAAGGCG CAGCGGTCGG GCTGAACGGG GGGTTCGTGC ACACAGCCCA GCTTGGAGCG 3901 AACGACCTAC ACCGAACTGA GATACCTACA GCGTGAGCAT TGAGAAAGCG CCACGCTTCC 3961 CGAAGGGAGA AAGGCGGACA GGTATCCGGT AAGCGGCAGG GTCGGAACAG GAGAGCGCAC 4021 GAGGGAGCTT CCAGGGGGAA ACGCCTGGTA TCTTTATAGT CCTGTCGGGT TTCGCCACCT 4081 CTGACTTGAG CGTCGATTTT TGTGATGCTC GTCAGGGGGG CGGAGCCTAT GGAAAAACGC 4141 CAGCAACGCG GCCTTTTTAC GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC ACATGTTCTT 4201 TCCTGCGTTA TCCCCTGATT CTGTGGATAA CCGTATTACC GCCTTTGAGT GAGCTGATAC 4261 CGCTCGCCGC AGCCGAACGA CCGAGCGCAG CGAGTCAGTG AGCGAGGAAG CGGAAGAGCG 4321 CCCAATACGC AAACCGCCTC TCCCCGCGCG TTGGCCGATT CATTAATGCA GCTGGCACGA 4381 CAGGTTTCCC GACTGGAAAG CGGGCAGTGA GCGCAACGCA ATTAATGTGA GTTAGCTCAC 4441 TCATTAGGCA CCCCAGGCTT TACACTTTAT GCTTCCGGCT CGTATGTTGT GTGGAATTGT 4501 GAGCGGATAA CAATTTCACA CAGGAAACAG CTATGACCAT GATTACGCCA AGCTgtaagt 4561 ttaaacatga tcttactaac taactattct catttaaatt ttcagAGCTT AAAAATGGCT 4621 GAAATCACTC ACAACGATGG ATACGCTAAC AACTTGGAAA TGAAAT //