LOCUS       mtk0_045                6616 bp ds-DNA     circular     22-FEB-2019
DEFINITION  .
FEATURES             Location/Qualifiers
     misc_feature    1017..1159
                     /label="ConE'"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     misc_feature    3821..3821
                     /label="mutation to remove BsaI cut site"
                     /ApEinfo_revcolor=#ff9ccd
                     /ApEinfo_fwdcolor=#ff9ccd
     protein_bind    complement(6459..6462)
                     /label="BsmBI"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     misc_feature    3934..3934
                     /label="mutation to reduce GC content"
                     /ApEinfo_revcolor=#b7e6d7
                     /ApEinfo_fwdcolor=#b7e6d7
     promoter        1831..2356
                     /label="CMV promoter"
                     /ApEinfo_revcolor=#c7b0e3
                     /ApEinfo_fwdcolor=#c7b0e3
     CDS             2368..3390
                     /label="HPH (Hygromycin Phosphotransferase)"
                     /ApEinfo_revcolor=#9eafd2
                     /ApEinfo_fwdcolor=#9eafd2
     RBS             131..166
                     /label="sfGFP Ribosome Binding Site"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     misc_feature    3968..3968
                     /label="mutation to reduce GC"
                     /ApEinfo_revcolor=#d6b295
                     /ApEinfo_fwdcolor=#d6b295
     CDS             167..883
                     /label="sfGFP"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     primer_bind     4224..4240
                     /label="M13 Forward (-40) (Elim)"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    1167..1191
                     /label="ConE scar"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     CDS             complement(4280..5095)
                     /label="KanR"
                     /ApEinfo_revcolor=#008000
                     /ApEinfo_fwdcolor=#008000
     promoter        complement(5096..5243)
                     /label="KanR Promoter"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     misc_feature    6470..6612
                     /label="ConS'"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     protein_bind    1167..1170
                     /label="BsmBI"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     terminator      884..1012
                     /label="BBa_B0015 Terminator"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     protein_bind    1160..1165
                     /label="BsmBI"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     misc_feature    1221..1500
                     /label="HS4"
                     /ApEinfo_revcolor=#ff9ccd
                     /ApEinfo_fwdcolor=#ff9ccd
     misc_feature    4006..4006
                     /label="mutation to reduce GC content"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     protein_bind    complement(6464..6469)
                     /label="BsmBI"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     misc_feature    4003..4003
                     /label="mutation to remove ngg"
                     /ApEinfo_revcolor=#b7e6d7
                     /ApEinfo_fwdcolor=#b7e6d7
     misc_feature    3977..3977
                     /label="mutation to reduce GC"
                     /ApEinfo_revcolor=#d6b295
                     /ApEinfo_fwdcolor=#d6b295
     misc_feature    4080..4080
                     /label="mutation to reduce GC"
                     /ApEinfo_revcolor=#b4abac
                     /ApEinfo_fwdcolor=#b4abac
     misc_feature    3638..4223
                     /label="Right Arm Homology"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    3904..3904
                     /label="mutation to remove BsmBI cut site"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     misc_feature    4095..4095
                     /label="mutation to remove ngg"
                     /ApEinfo_revcolor=#9eafd2
                     /ApEinfo_fwdcolor=#9eafd2
     misc_feature    4086..4086
                     /label="mutation to reduce GC"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     promoter        1..130
                     /label="BBa_J72163 GlpT Promoter"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     misc_feature    1504..1783
                     /label="HS4"
                     /ApEinfo_revcolor=#f58a5e
                     /ApEinfo_fwdcolor=#f58a5e
     misc_feature    complement(4097..4116)
                     /label="gRNA"
                     /ApEinfo_revcolor=#f8d3a9
                     /ApEinfo_fwdcolor=#f8d3a9
     Terminator      3400..3633
                     /label="BGH-PA"
                     /ApEinfo_revcolor=#c7b0e3
                     /ApEinfo_fwdcolor=#c7b0e3
     misc_feature    6039..6439
                     /label="Left Arm"
                     /ApEinfo_revcolor=#75c6a9
                     /ApEinfo_fwdcolor=#75c6a9
     promoter        complement(6020..6038)
                     /label="M13_reverse_primer"
                     /ApEinfo_revcolor=#ff9ccd
                     /ApEinfo_fwdcolor=#ff9ccd
     misc_feature    2359..2367
                     /label="kozac"
                     /ApEinfo_revcolor=#d6b295
                     /ApEinfo_fwdcolor=#d6b295
     terminator      complement(4253..4279)
                     /label="KanR Terminator"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     rep_origin      complement(5244..6007)
                     /label="ColE1"
                     /ApEinfo_revcolor=#7f7f7f
                     /ApEinfo_fwdcolor=#7f7f7f
     misc_feature    complement(3878..3897)
                     /label="gRNA1"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     misc_feature    3876..3876
                     /label="mutation to change ngg"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
ORIGIN
        1 gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt gtaaatctta tttaataatg
       61 tgtgcggcaa ttcacattta atttatgaat gttttcttaa catcgcggca actcaagaaa
      121 cggcaggttc ggatcttagc tactagagaa agaggagaaa tactagatgc gtaaaggcga
      181 agagctgttc actggtgtcg tccctattct ggtggaactg gatggtgatg tcaacggtca
      241 taagttttcc gtgcgtggcg agggtgaagg tgacgcaact aatggtaaac tgacgctgaa
      301 gttcatctgt actactggta aactgccggt tccttggccg actctggtaa cgacgctgac
      361 ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg aagcagcatg acttcttcaa
      421 gtccgccatg ccggaaggct atgtgcagga acgcacgatt tcctttaagg atgacggcac
      481 gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc ctggtaaacc gcattgagct
      541 gaaaggcatt gactttaaag aggacggcaa tatcctgggc cataagctgg aatacaattt
      601 taacagccac aatgtttaca tcaccgccga taaacaaaaa aatggcatta aagcgaattt
      661 taaaattcgc cacaacgtgg aggatggcag cgtgcagctg gctgatcact accagcaaaa
      721 cactccaatc ggtgatggtc ctgttctgct gccagacaat cactatctga gcacgcaaag
      781 cgttctgtct aaagatccga acgagaaacg cgatcatatg gttctgctgg agttcgtaac
      841 cgcagcgggc atcacgcatg gtatggatga actgtacaaa tgaccaggca tcaaataaaa
      901 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
      961 ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tagctggaaa
     1021 tctgctcgtc agtggtgctc acactgacga atcatgtaca gatcataccg atgactgcct
     1081 ggcgactcac aactaagcaa gacagccgga accagcgccg gcgaacacca ctgcatatat
     1141 ggcatatcac aacagtccac gtctcaagca gttacagaga tgttacgaac cactagtgca
     1201 ctgcagATCG AATTCGAGTT GGCGCGCCTG TCATTCTAAA TCTCTCTTTC AGCCTAAAGC
     1261 TTTTTCCCCG TATCCCCCCA GGTGTCTGCA GGCTCAAAGA GCAGCGAGAA GCGTTCAGAG
     1321 GAAAGCGATC CCGTGCCACC TTCCCCGTGC CCGGGCTGTC CCCGCACGCT GCCGGCTCGG
     1381 GGATGCGGGG GGAGCGCCGG ACCGGAGCGG AGCCCCGGGC GGCTCGCTGC TGCCCCCTAG
     1441 CGGGGGAGGG ACGTAATTAC ATCCCTGGGG GCTTTGGGAG GGGGCTGTCC CCGTGAGCTC
     1501 CCAGGCGCGC CTGTCATTCT AAATCTCTCT TTCAGCCTAA AGCTTTTTCC CCGTATCCCC
     1561 CCAGGTGTCT GCAGGCTCAA AGAGCAGCGA GAAGCGTTCA GAGGAAAGCG ATCCCGTGCC
     1621 ACCTTCCCCG TGCCCGGGCT GTCCCCGCAC GCTGCCGGCT CGGGGATGCG GGGGGAGCGC
     1681 CGGACCGGAG CGGAGCCCCG GGCGGCTCGC TGCTGCCCCC TAGCGGGGGA GGGACGTAAT
     1741 TACATCCCTG GGGGCTTTGG GGGGGGGCTG TCCCCGTGAG CTCAATAAAA GAGCCCACta
     1801 caGCGGTGGG CTCTATGGGG TACCTACGTA ggtacataac ttacggtaaa tggcccgcct
     1861 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     1921 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     1981 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     2041 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     2101 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     2161 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     2221 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     2281 ccattgaCGC AAATGGGCGG TAGGCGTGta cggtgggagg tctatataag cagagctcgt
     2341 ttagtgaacc gtcagataGC CACCATGggt aaaaagcctg aactcaccgc gacgtctgtc
     2401 gagaagtttc tgatcgaaaa gttcgacagc gtgtccgacc tgatgcagct ctcggagggc
     2461 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     2521 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     2581 ctcccgattc cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc
     2641 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     2701 ctgcaaccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     2761 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     2821 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     2881 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     2941 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     3001 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     3061 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     3121 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     3181 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     3241 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     3301 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     3361 cgccccagca ctcgtccgag ggcaaaggaa tAACTCGAGC GACGCCTCGA CTGTGCCTTC
     3421 TAGTTGCCAG CCATCTGTTG TTTGCCCCTC CCCCGTGCCT TCCTTGACCC TGGAAGGTGC
     3481 CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC TGAGTAGGTG
     3541 TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG GGGGAGGATT GGGAAGACAA
     3601 TAGCAGGCAT GCTGGGGATG CGGTGGGCTC TATGAGTAGA TCgcgggGGA GGGAGGGaTT
     3661 CTTCtAGGCC CAGGGCGGTC CTTAggAGAC aGGAGGCAGC cGAGAACTCC CATAAAGGTA
     3721 TTGCGGCACT CCCCTCCCCC TaCCgAGAAG GGTGCGGCCT TCTCTCCgCC TCCTCCACtG
     3781 CAGaTCCCTC AGGATTGCAG CTCGCGCCGa TTTTcaGAGA TCAcGCaCCT CCCtCCCACA
     3841 AACCAGCaGG ACCGACCCCC GCTCCTCCCC CcACCTCCgC GAGTccgcga gcggatccag
     3901 ctcATCTCGt CCTTCAGGCG GcGGacgCCC gGGACGGAGC CgCGGtCGCC GCCAcCCAGA
     3961 AGcCTCGACC caGCAGTCCG cctccgcctc cgcctccgCC TCTAGTGCGT GCGCAGGGGC
     4021 GCGGGGcCAG ACACTGCGGC GCggGGCcTC GGGaAGGACC GTACCAACGa CCGCCTCCCT
     4081 gCCCCTCCGC cCCCTctgct gacccGCtCA GTGGTTTCGC TCAattggga cccgagccaa
     4141 tggcaagggg ccgttccccg gcttcagcag ccaatcaggt gccgaagcca cacgggcggc
     4201 gggtaaccct actccccgcg agggttttcc cagtcacgac CCGAgcggcc gcgtgttaca
     4261 accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
     4321 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     4381 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     4441 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     4501 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     4561 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     4621 cgttattcat tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac
     4681 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     4741 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     4801 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     4861 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     4921 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     4981 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     5041 tgttggaatt taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac
     5101 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     5161 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa
     5221 cttttgctga gttgaaggat cagtcatgac caaaatccct taacgtgagt tttcgttcca
     5281 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5341 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5401 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5461 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5521 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5581 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5641 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5701 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5761 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5821 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5881 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5941 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6001 taaccgtgcg gccgccaatc atggtcatag ctgtttccTG CAAcggaaag ggcCTAAAAT
     6061 TTTAAGATTa gtatctaatt taaaATGCTA CACTTTGGTG GTGtaaacaa cgttctcaaa
     6121 cgacgtgagt aaaaaactca gatgaccatg cccttaacct ccttttatcc acaggaaaag
     6181 cactacaaaa acgatcttaa gtagtaataa cacgcagcct tgcTGCATAA AATCAGCccg
     6241 agatgaatgc ctggactcCC ATTTCCCTcC tCCACCCcTA TTAtaattat tttgtgacac
     6301 aaactcaagc agggtgaaga gagaaatctc acacCTGCTC AaTCCCCTGC AGGaCAACGC
     6361 CCAGacacca ggtaaagcct gggcacagag agTCCCGCCC AcAATCGAGA AGgagCaACT
     6421 CGACATGGAG GCGATGACGC CCTgaattcg catctagact gatgagacgt ggtagagcca
     6481 caaacagccg gtacaagcaa cgatctccag gaccatctga atcatgcgcg gatgacacga
     6541 actcacgacg gcgatcacag acattaaccc acagtacaga cactgcgaca acgtggcaat
     6601 tcgtcgcaat acaacg
//