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 
//
