LOCUS       Exported                9554 bp ds-DNA     circular SYN 16-JAN-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9554)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jan 17, 2020 from SnapGene Viewer 5.0.6
            https://www.snapgene.com
COMMENT     Add mScarlet (use this on chimeric WT, chimeric D290V, and CE pBS)
            Amplify pBS
            mS_pBS_R (need to reverse 
            complement):CGTGAAACCCGTGGCGGAAGATCTCGCGATATGGTGAGCAAGGGCG
            mS_pBS_F: GACGAGCTGTACAAGGGACAGCGTGGCGGTTACAATGGCGGTGGTG
            
            Amplify mScarlet:
            mScarlet_F: GGAAGATCTCGCGATATGGTGAGCAAGGGCGAGGCAGTGATCAAGG
            mScarlet_R (need to reverse complement): 
            CTCCACCGGCGGCATGGACGAGCTGTACAAGGGACAGCGTGGCGGT
FEATURES             Location/Qualifiers
     source          1..9554
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..307
                     /locus_tag="F1 Ori"
                     /label=F1 Ori
     misc_feature    326..394
                     /locus_tag="lacz a"
                     /label=lacz a
     misc_feature    465..482
                     /locus_tag="M13F"
                     /label=M13F
     misc_feature    524..543
                     /locus_tag="5' homology fwd "
                     /label=5' homology fwd
     misc_feature    535..543
                     /locus_tag="NotI"
                     /label=NotI
     misc_feature    543
                     /locus_tag="XbaI"
                     /label=XbaI
     misc_feature    544..564
                     /locus_tag="F primer"
                     /label=F primer
     misc_feature    544..561
                     /locus_tag="Beginning of next 5' gene(1)"
                     /label=Beginning of next 5' gene
     misc_feature    562..569
                     /locus_tag="Beginning of next 5' gene"
                     /label=Beginning of next 5' gene
     misc_feature    1168..1187
                     /locus_tag="hrp-1_pro_1F"
                     /label=hrp-1_pro_1F
     misc_feature    1909..1929
                     /locus_tag="hrp-1_pro_2F"
                     /label=hrp-1_pro_2F
     misc_feature    2656..2678
                     /locus_tag="hrp-1_pro_3F"
                     /label=hrp-1_pro_3F
     misc_feature    3383..3409
                     /locus_tag="hrp-1_pro_4F"
                     /label=hrp-1_pro_4F
     misc_feature    3830..3856
                     /locus_tag="Seq_primer_stop_exon1/2 B9 CRISPR box"
                     /label=Seq_primer_stop_exon1/2 B9 CRISPR box
     misc_feature    3920..4136
                     /locus_tag="5' UTR"
                     /label=5' UTR
     misc_feature    3921..3940
                     /locus_tag="hrp-1_5'_1F"
                     /label=hrp-1_5'_1F
     misc_feature    4059..4085
                     /locus_tag="Exon1"
                     /label=Exon1
     misc_feature    4130..4136
                     /locus_tag="SanDIII/PpumI"
                     /label=SanDIII/PpumI
     misc_feature    4137..4777
                     /locus_tag="5' Homology arm"
                     /label=5' Homology arm
     misc_feature    4166..4777
                     /locus_tag="Exon2"
                     /label=Exon2
     misc_feature    4203..4208
                     /locus_tag="BstbI"
                     /label=BstbI
     misc_feature    4624..4643
                     /locus_tag="seqprimer1"
                     /label=seqprimer1
     misc_feature    4748..4793
                     /locus_tag="mS_pBS_R"
                     /label=mS_pBS_R
     misc_feature    4763..4808
                     /locus_tag="mScarlet_F"
                     /label=mScarlet_F
     misc_feature    4771..4776
                     /locus_tag="NruI"
                     /label=NruI
     misc_feature    4778..5473
                     /locus_tag="mScarlet"
                     /label=mScarlet
     misc_feature    5443..5488
                     /locus_tag="mScarlet_R"
                     /label=mScarlet_R
     misc_feature    5459..5504
                     /locus_tag="mS_pBS_F"
                     /label=mS_pBS_F
     misc_feature    5474..5651
                     /locus_tag="5' Homology arm(1)"
                     /label=5' Homology arm
     misc_feature    5474..5597
                     /locus_tag="Exon2(1)"
                     /label=Exon2
     misc_feature    5474..5597
                     /locus_tag="LC in exon 2"
                     /label=LC in exon 2
     misc_feature    5611..5630
                     /locus_tag="repl_seq_1F"
                     /label=repl_seq_1F
     misc_feature    5625..5651
                     /locus_tag="5' homology rev"
                     /label=5' homology rev
     misc_feature    5652..5927
                     /locus_tag="optimized HS LC"
                     /label=optimized HS LC
     misc_feature    5772..5774
                     /locus_tag="D to V"
                     /label=D to V
     misc_feature    5773
                     /locus_tag="AtoT (HS mutation, most used Val codon)"
                     /label=AtoT (HS mutation, most used Val codon)
     misc_feature    5928..7147
                     /locus_tag="3' Homology arm"
                     /label=3' Homology arm
     misc_feature    5928..5958
                     /locus_tag="3' homology fwd"
                     /label=3' homology fwd
     misc_feature    5944..5965
                     /locus_tag="hrp-1_repl_seq_R"
                     /label=hrp-1_repl_seq_R
     misc_feature    6123..6142
                     /locus_tag="seqprimer2"
                     /label=seqprimer2
     misc_feature    7116..7174
                     /locus_tag="3' homology rev"
                     /label=3' homology rev
     misc_feature    7153
                     /locus_tag="BamHI"
                     /label=BamHI
     misc_feature    7267..7287
                     /locus_tag="M13R"
                     /label=M13R
     misc_feature    7293..7315
                     /locus_tag="LacO"
                     /label=LacO
     misc_feature    7655..8337
                     /locus_tag="ColE1 Ori"
                     /label=ColE1 Ori
     misc_feature    8435..9094
                     /locus_tag="AmpR"
                     /label=AmpR
ORIGIN
        1 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
       61 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
      121 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
      181 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
      241 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
      301 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
      361 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      421 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      481 gtgagcgcgc gtaatacgac tcactatagg gcgaattgga gctccaccgc ggtggcggcc
      541 gctgtaggag ctgagcccat cttcaatgcc tccgattttc caaaattaaa cactttttcc
      601 atgaattctt ccattaattt ggcaattgag ccaaatgaaa agccagaaga gccagatgga
      661 gccggtgttg gagttggagc agacagagtt gaacggagtt ctggagcagc tggggttggg
      721 gtcggagcag caagagttga acgtggttca gggtcggcga ggaactgaaa atctaggttt
      781 tttaggaaaa attcgcctaa aaacatgccg aaaaaatttc cgaattttag acaattctgg
      841 aaaaaattgt agtgaaattg tctaaaaatt ttggtaaaag tttgcagaaa actgtcagaa
      901 aatgttaatt tcttacgtgt ttccttgctt gggacactag aacagcaccg aataagacaa
      961 aaagtacaaa acacagcttc atgttggaat ttatgccaga aaaatagctg taactcggct
     1021 tttatagaat agaattggtt cctggtgatg ctggagttct attaaacatg agaacaggag
     1081 tatgaaccgt gggaagtggt gttttggttg gagaggtgtt ctgcacggct cggcaattgt
     1141 ggtgcatcag tgtccaccaa acatcaagca taacctggat gataaggaga caagctttgg
     1201 tgcaaaagtt ttcaaatttt aaagcaattt ttaggactct tcaaagcatg aaactgtttc
     1261 tcaaggtttc cttctacttc taactccttc tagatgcatc acggcctcaa aatgtaaaac
     1321 ctcagaaatg gtaccagagg ttcaaaaatg caaatatcag ggacctgggc tagatcttct
     1381 cgtttcgagg gctgggaaac tgcccttccg tgtgaaaggg gcaattgtca ggcctggagc
     1441 atgatattga tcggttttgt gagaagacca acaaggtagc taataaatcg atcctatatt
     1501 gtattaacac atgtatttga atcagatctt tcatttctca ttgactccct caaatttcca
     1561 gcaagaatgg atgctacaat tgtaagttta accacaaatc atttttaatt atacctgaat
     1621 ttcttttcag accaaaacca gaaaagtcgt attcatatag tgttgttcaa gagattgtca
     1681 gttgtatgtg cctaacaaaa aaaaatctat ggcattctaa atttacaatt tcagcatagt
     1741 cgctcgccat gtgtcaccac tctttcgcca aaccatcgat ttgatggatc caggcattga
     1801 gaaaattcat ttactcagtt ctgccagtcc gtagtgtagt ttttaccgga tatagagaat
     1861 tgaatctaga aaacctggct gcctggcaag aagtcactgg tagtaacagg ctgatgaggg
     1921 cagccttcgg tgagctatcc gacgtgatgg gcatgccgaa acttagacta agaaagttca
     1981 gttttgataa cacatatgga ctggttgggt tatctgggaa agagaatatg caatctgtgg
     2041 atgactgctt caaggctatg aacttaccac atgcgaaggt gcttgaagtg actggaatcg
     2101 actttgtgga caatatttcg atggtgaagt actttcgccc aggttatctc gagaaaatcc
     2161 gcatgtattc ttgttattgc tcagctagcc aggagatgag tgaagaaatt gcaaatctcg
     2221 agcagtggaa gcaagcgaaa cattttctgt gcattggtgg tagaatgaac attcctattc
     2281 aaagattttt ccactttgac cagttctcca tcggtcttca gactctatca atcgatgatg
     2341 ctgttcagat aagagatgtg agttggtagt taaagtagcg ttttgtaatg tattctatat
     2401 tccagattct cctcagatcg cccagcttcg agtcagcaac tatcgatgtt caactacttg
     2461 aaaaagaagc cattgagaac gtctttacgc cgaatcacct attaatttat agaggaagag
     2521 agttcagacc ggtgggaact gattccagtt ttgtgatcaa tataacaaaa aacaagcttg
     2581 aaatccacaa gcacaaggga gctaatccac cacgtcgtag agacgaacgg ccaatccacg
     2641 agcgtatacg tcaaaggcaa gatggatggt gaagtttcaa tggttgctct aatacgttta
     2701 catgtctatc aaaccttttt ctgttactta agtaaatcgt tgtttcatat ttaaccagtc
     2761 ttacctacgg ctggcatatc ggacgtccgg cgtcagaacc ttgtgcgtct ggttttagat
     2821 gtttgaaaat tttggaaaat tattaaagtc tattgataac ctgtcaaaac cggacgaccg
     2881 gacgccaaat atgacagccc tagtcttacc agtataatat ttttataaac ctaaaacagg
     2941 tatgaatatc tcttttttct gcgcagcgct gggatgcttc tgtttcagaa aaatcacatg
     3001 gtataaaaaa tttccaaatt acagtagtcc taccgtaatc ctgcattatc ccactactgt
     3061 atgtaccttg acattatttt tcctcgccca actcccagcc caatatatct cttcaacagc
     3121 taagaactac atttttccta aactacagta aaccaacagt atccctacag taccacatta
     3181 attataaatt aaaacattat ttttcataag aaatcgtgag aatttcgaga aaaataaagg
     3241 attcaaagat tatttgaacg tttttttcgt gaaaaaatgt caaagaaatc aaaatgttca
     3301 tcttccttgt gaaaaatccg aaaagccatt ataaactagt tgattttcat aaaaatttgt
     3361 agaaatattt ttcttaaaat ttgtagaaga attttaacgt tttttaaaca aaaattccaa
     3421 aattggaatt tggtattgaa aatacttttt aaaagttatt tttttaattt gtaaaaattt
     3481 aactaaattc aaaaaataaa gtttccctcc tttttttctt aaaggcacac acatttatcg
     3541 actcgggtct cgccgcgaac agacgcgacg cgacgcggtt aggaatgcgg cgcgtcgcgt
     3601 gtaacccttt tctctgtggt tttcgagagc attctctctc ggttttcgtc acaaaaaggt
     3661 atgttttaca ggttttccgc gcactgtttt cgcgtaattc ttcattttaa ttcagataat
     3721 ctaattgttg tgtagtttta gctcgaattt atccgtaatc tttgattaaa tacgaataaa
     3781 actcgctaaa atagtcttat ttctcagata tttatcgatt tgttattttc cacgaatagt
     3841 ctactcgtgc atggcgtcta tatttaactt aataatctat ttttagattt atctttactc
     3901 taacttgtac ctatttcagt gatcaacacc tctgtcactc tttgtgagta atagcgttcg
     3961 ataggtagtt acaccgtgaa actagcctgt ctatttccta cttcataagc taagaactac
     4021 ttgttagcac aatccgatcg actactaaga actagatcat gacggacgtg gaaatcaagg
     4081 ctgaggttac gtttgttcac ttgcttttag ggtgtaggcc acctgatttg ggtcccgatc
     4141 atttaattat cgattttatt ttcagaatgg aagcggagat gcctcgctcg agccagagaa
     4201 ccttcgaaag attttcgttg gaggactcac gtcaaacacc accgatgacc tgatgcgaga
     4261 gttctactca caattcggag aaatcactga tatcatcgtc atgagagatc caactaccaa
     4321 gagatcccgc ggattcggat ttgtcacctt ctctggcaaa actgaagtcg atgctgccat
     4381 gaaacaacgc ccgcacatca tcgacggaaa gaccgtggac ccgaagcgtg ccgtgccacg
     4441 tgatgataag aaccgctccg agtcgaatgt ctccaccaaa cgcctgtacg tcagcggagt
     4501 tcgtgaggat cacaccgagg acatgctcac cgagtatttc acaaagtatg gaaccgtcac
     4561 caaatccgag attattctcg acaaagccac ccaaaagcca agaggcttcg gattcgtcac
     4621 cttcgatgat catgactctg tggatcaatg cgttcttcaa aaatcccata tggtcaacgg
     4681 acacagatgt gacgtgcgca aaggactctc caaggacgag atgagcaagg ctcagatgaa
     4741 ccgcgatcgt gaaacccgtg gcggaagatc tcgcgatatg gtgagcaagg gcgaggcagt
     4801 gatcaaggag ttcatgcggt tcaaggtgca catggagggc tccatgaacg gccacgagtt
     4861 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     4921 ggtgaccaag ggtggccccc tgcccttctc ctgggacatc ctgtcccctc agttcatgta
     4981 cggctccagg gccttcacca agcaccccgc cgacatcccc gactactata agcagtcctt
     5041 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt
     5101 gacccaggac acctccctgg aggacggcac cctgatctac aaggtgaagc tccgcggcac
     5161 caacttccct cctgacggcc ccgtaatgca gaagaagaca atgggctggg aagcgtccac
     5221 cgagcggttg taccccgagg acggcgtgct gaagggcgac attaagatgg ccctgcgcct
     5281 gaaggacggc ggccgctacc tggcggactt caagaccacc tacaaggcca agaagcccgt
     5341 gcagatgccc ggcgcctaca acgtcgaccg caagttggac atcacctccc acaacgagga
     5401 ctacaccgtg gtggaacagt acgaacgctc cgagggccgc cactccaccg gcggcatgga
     5461 cgagctgtac aagggacagc gtggcggtta caatggcggt ggtggtggcg gcggcggctg
     5521 gggaggacca gctcaacgcg gcggtccagg agcatatggt ggaccaggag gtggcggcca
     5581 aggaggatat ggcggaggta gttggattct ggcttgtgaa ttctcgattc taatattatt
     5641 aactttcaga ttacggagga ggacgcggtg gttatggtgg tggaggacca ggatatggaa
     5701 atcagggtgg aggatatgga ggaggatatg ataattatgg aggaggaaat tacggatctg
     5761 gaaattacaa tgatttcgga aactacaacc agcagccatc taattatgga ccaatgaagt
     5821 ctggaaattt cggaggatct cgcaatatgg gtggaccata tggaggagga aattatggtc
     5881 caggaggatc tggaggatct ggaggatatg gaggacgttc tcgttactaa attaattcct
     5941 taagcccctc taagtgtcca acgtgtctga gcttcccgtg ctctctcctc tgtatcgatc
     6001 ttctcaacca tttttgctct gctatctcat caaaatcttc tgtacttttt cttgttgcct
     6061 ttctccagaa agtttcgttt attattcaat tttttaccct gtctttttta tataatttat
     6121 ttctaaattc gcacagttga catccattca actagagaat gaatgcagag tttgacacca
     6181 aattggctta attttcaagc tgtttttaac caaaataata aaaaagtctc aactttacaa
     6241 aaatgtttat ctggatctgg acgacagcga gctgagaaat tgatatattt tcagagtaaa
     6301 aactaaaaat gtctggaatt tttggagtca ttgtctcagg gaggacgccg atcgaagtgg
     6361 tgccggtctc cgatacggag ttcacctgcg aaattgtaaa cgctgacgcg ataaaccacg
     6421 tggtggtctt ccttaccgga gctcaaccgt ttccggacgg aattggtggc tctggtgatc
     6481 attttcagaa ttttccgccc aaaaaaaccc aatttatttc agtttacatc agatggccca
     6541 cccaagacgg cggaaattgg cattatttgg gatttatttg caacgagaag cccagcgcga
     6601 tttataaagt tgctcaggtg tgcatttttg atggaaaaat gaaagaaaac caccgaaatt
     6661 tcgatgtttc tctgtcgatt ttttttattt aaaaacttag aaatactctc aatttcgcat
     6721 ttttcaccaa aaaaaagaat ctcaactggt ttttcacata gcttaatttt tgaagaaaaa
     6781 tctcaacaaa acccagtttt aagtttcaaa ttctcaaaaa ttgatctttt ttttccctta
     6841 aaaatccagt ttttagctcc aaattgtcat aattttccag aaaaatttag ttttcgagct
     6901 aacaacatta aaatttcaca gaaaatctga atttcagcct tagagatcca aatttttcgg
     6961 ctgaaaatca caatttttcc atttttcagc tccacaaatc cgacgcttct cactcgtttt
     7021 cccaatttga caatcaaatg caactatatt ccagtggatc agctcaaatt ggaattaatg
     7081 ctgaaagttt atctgatatc actggacggc aggcagctga tgggacacag gctagccagc
     7141 aggtgaagga tcccccgggc tgcaggaatt cgatatcaag cttatcgata ccgtcgacct
     7201 cgaggggggg cccggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     7261 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     7321 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     7381 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     7441 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     7501 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     7561 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     7621 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     7681 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     7741 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     7801 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     7861 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     7921 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     7981 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     8041 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     8101 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     8161 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     8221 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     8281 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     8341 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     8401 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     8461 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     8521 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     8581 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     8641 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     8701 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     8761 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     8821 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     8881 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     8941 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     9001 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     9061 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     9121 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     9181 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     9241 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     9301 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     9361 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     9421 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     9481 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     9541 gatagggttg agtg
//
