LOCUS       pPRISM_Stop_cmlc2_eGFP        6145 bp ds-DNA     circular     11-SEP-2018
DEFINITION  synthetic circular DNA, gene-targeting cassette
ACCESSION   .
VERSION     2.1
KEYWORDS    pPRISM, stop
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6145)
  AUTHORS   Jordan M Welker,  Maira P Almeida, Melanie Torrie, Wesley A Wierson, Carla M Mann, Trevor Weiss, Cassandra Bullard, Katherine Knudtson, Kenna McKeighan, Stephen C. Ekker, Drena L Dobbs, Maura McGrail, Karl J Clark, Jeffrey J Essner
  TITLE     pPRISM an expanded tool set for precise genome editing
  JOURNAL   .
COMMENT     3x3 Stop sequence, was designed to have three stop condons in each
            frame in theoretically optimal context     
COMMENT     
COMMENT     ApEinfo:methylated:0
FEATURES             Location/Qualifiers
     misc_feature    931..963
                     /locus_tag="3x3 Stop sequence"
                     /label="3x3 Stop sequence"
                     /ApEinfo_label="3x3 Stop sequence"
                     /ApEinfo_fwdcolor="#8080c0"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(267..457)
                     /note="ITR(R)"
                     /note="Tol2 ITR(R)"
                     /locus_tag="ITR(R)"
                     /label="ITR(R)"
                     /ApEinfo_label="ITR(R)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="#80ffff"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    918..923
                     /locus_tag="BfuAI site 2"
                     /label="BfuAI site 2"
                     /ApEinfo_label="BfuAI site 2"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    4396..4610
                     /note="ITR(L)"
                     /note="Tol2 ITR(L)"
                     /locus_tag="ITR(L)"
                     /label="ITR(L)"
                     /ApEinfo_label="ITR(L)"
                     /ApEinfo_fwdcolor="#80ffff"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             4675..5484
                     /note="Kan(R)"
                     /locus_tag="Kan(R)"
                     /label="Kan(R)"
                     /ApEinfo_label="Kan(R)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    928..931
                     /locus_tag="BfuAI site 2 overhang"
                     /label="BfuAI site 2 overhang"
                     /ApEinfo_label="BfuAI site 2 overhang"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     rep_origin      complement(5517..6145)
                     /direction=LEFT
                     /note="ColE1 Ori"
                     /locus_tag="ColE1 Ori"
                     /label="ColE1 Ori"
                     /ApEinfo_label="ColE1 Ori"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(514..519)
                     /locus_tag="BfuAI site 1"
                     /label="BfuAI site 1"
                     /ApEinfo_label="BfuAI site 1"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     rep_origin      complement(1..253)
                     /direction=LEFT
                     /note="ColE1 Ori"
                     /locus_tag="ColE1 Ori(1)"
                     /label="ColE1 Ori(1)"
                     /ApEinfo_label="ColE1 Ori"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     intron          2492..2698
                     /note="mIntron"
                     /note="carp bactin shortened intron I"
                     /locus_tag="mIntron"
                     /label="mIntron"
                     /ApEinfo_label="mIntron"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     polyA_signal    3939..4143
                     /note="SV40(A)"
                     /note="SV40"
                     /locus_tag="SV40(A)"
                     /label="SV40(A)"
                     /ApEinfo_label="SV40(A)"
                     /ApEinfo_fwdcolor="#ff80c0"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    487..509
                     /locus_tag="5' Universal Guide (w/PAM)"
                     /label="5' Universal Guide (w/PAM)"
                     /ApEinfo_label="5' Universal Guide (w/PAM)"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    642..917
                     /vntifkey="21"
                     /locus_tag="lacZ'"
                     /label="lacZ'"
                     /ApEinfo_label="lacZ'"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="#1dda7b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        520..641
                     /vntifkey="30"
                     /locus_tag="lacZ\promoter"
                     /label="lacZ\promoter"
                     /ApEinfo_label="lacZ\promoter"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(506..509)
                     /locus_tag="BfuAI site 1 overhang"
                     /label="BfuAI site 1 overhang"
                     /ApEinfo_label="BfuAI site 1 overhang"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(4332..4354)
                     /locus_tag="3' Universal Guide (w/PAM)"
                     /label="3' Universal Guide (w/PAM)"
                     /ApEinfo_label="3' Universal Guide (w/PAM)"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     polyA_signal    971..2146
                     /note="ocean pout terminator"
                     /note="3'UTR, poly(A) signal, and putative boundary
                     element from ocean pout (FRM- Pat Gibbs)."
                     /locus_tag="ocean pout terminator"
                     /label="ocean pout terminator"
                     /ApEinfo_label="ocean pout terminator"
                     /ApEinfo_fwdcolor="#ff8000"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     intron          4233..4321
                     /note="Carp beta-actin intron 1 filler"
                     /locus_tag="Carp beta-actin intron 1 filler"
                     /label="Carp beta-actin intron 1 filler"
                     /ApEinfo_label="Carp beta-actin intron 1 filler"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     polyA_signal    3473..3697
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
                     /locus_tag="bGH poly(A) signal"
                     /label="bGH poly(A) signal"
                     /ApEinfo_label="bGH poly(A) signal"
                     /ApEinfo_fwdcolor="#8080ff"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    4324..4330
                     /locus_tag="BspQI site 2"
                     /label="BspQI site 2"
                     /ApEinfo_label="BspQI site 2"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     polyA_signal    3734..3938
                     /note="SV40(A)"
                     /note="SV40"
                     /locus_tag="SV40(A)(1)"
                     /label="SV40(A)(1)"
                     /ApEinfo_label="SV40(A)"
                     /ApEinfo_fwdcolor="#ff80c0"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(4218..4224)
                     /locus_tag="BspQI site 1"
                     /label="BspQI site 1"
                     /ApEinfo_label="BspQI site 1"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    2173..2447
                     /note="cmlc2 enhancer/promoter xu lab"
                     /locus_tag="cmlc2 enhancer/promoter xu lab"
                     /label="cmlc2 enhancer/promoter xu lab"
                     /ApEinfo_label="cmlc2 enhancer/promoter xu lab"
                     /ApEinfo_fwdcolor="#ff80ff"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(4214..4216)
                     /locus_tag="BspQI site 1 overhang"
                     /label="BspQI site 1 overhang"
                     /ApEinfo_label="BspQI site 1 overhang"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_signal     2719..2748
                     /note="NLS"
                     /locus_tag="NLS"
                     /label="NLS"
                     /ApEinfo_label="NLS"
                     /ApEinfo_fwdcolor="#ffff80"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    4332..4334
                     /locus_tag="BspQI site 2 overhang"
                     /label="BspQI site 2 overhang"
                     /ApEinfo_label="BspQI site 2 overhang"
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             2749..3468
                     /vntifkey="4"
                     /locus_tag="eGFP"
                     /label="eGFP"
                     /ApEinfo_label="eGFP"
                     /ApEinfo_fwdcolor="#00ff0e"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
ORIGIN
        1 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
       61 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      241 ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc aaaaatttta
      301 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
      361 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
      421 aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta gtgagggtca
      481 attgatGGGA GGCGTTCGGG CCACAGCGGa cacgcaggtg cgcaacgcaa ttaatgtgag
      541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      661 gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg gggggcccgg
      721 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
      781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      901 gcctgaatgg cgaatggacc tgccacactt ctaTaaTgaT aggTagTaaT gatTagTaaT
      961 gagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct gtggagactg
     1021 ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc aaatttcctg
     1081 cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc tcactctgtg
     1141 tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt tagatgtgca
     1201 gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg acttggtgag
     1261 tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa ctgtgtgaat
     1321 ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt agtggaagtg
     1381 agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa tgcaaaaaaa
     1441 tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt attccaacac
     1501 gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt tttgtcaccc
     1561 tcacaggcaa tttagtataa tatattatat atatatatat atcatataat aatattcagt
     1621 ataatatata tatatatatc atattataat attcagtata atataaaaca caaacacata
     1681 tatgtataat ataatataac atttttattt attgagatgc ctctatggac cgtgttataa
     1741 gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat gttaaattaa
     1801 gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg cctccatgac
     1861 caagttaagc agatgagact gagacaactg tagaagacaa gatgttcact ttgctgaata
     1921 tagctggctt gacagttatc tatgactcta taaatatata tatatttttt tttttataaa
     1981 atgatttatt tataactata tatccatttc tcagacaggt gcttcatatc cctcactccc
     2041 gtagctgtcc atgctggatc tgtccccgtt gtttttaaaa agctaaataa gttattaaca
     2101 tgactgcatc cagcgagcca aacctgtctg gtgtacagct accagagaag cttgagatcc
     2161 tagtcaccgg cggcattcat ccatcctttt catccctcaa atctctcatt cacgtccccc
     2221 tccccatctg cacactttat ctcattttcc accctgctgg aatctgagca cttgtgcagt
     2281 tatcagggct cctgtattta ggaggctctg ggtgtccatg taggggacga acagaaacac
     2341 tgcagacctt tatagaagaa caaatgataa gagtcctcat acataaagac tccattagaa
     2401 acgtcagtga cccaggagcc cagaccaaca gcaaagcaga cagtgaagga tccacagcct
     2461 gactaactcg acaaaccccc ccaaacctaa ggtgagttga tctttaagct ttttacattt
     2521 tcagctcgca tatatcaatt cgcgcgcatt gtcacactag cacccactag cggtcagact
     2581 gcagaTTGCA GCAcGaaaca ggaagctgac tccacatggt cacatgctca ctgaagtgtt
     2641 gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta cagttcagcc
     2701 ggaattcacc tcgagaccat ggctccaaag aagaagcgta aggtaaccat ggtgagcaag
     2761 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2821 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2881 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2941 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     3001 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     3061 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3121 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     3181 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     3241 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     3301 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     3361 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3421 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag atctgtgcct
     3481 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3541 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3601 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3661 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccaaa
     3721 gggcgaatta attcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     3781 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     3841 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     3901 aggtttttta aagcaagtaa aacctctaca aatgtggtca ttgatgagtt tggacaaacc
     3961 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4021 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4081 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4141 ggtcattcag atcaattaac cctcacccgg gctagcgatg catgggagct ctgacgtacg
     4201 ttgtctagca aggaagtgaa gagctatccg gatatatatg tatgttatca taactgtact
     4261 ggctatgtca ggtggtaatg actgtaacgt tacgttactc gttgtaggca cgacattgaa
     4321 tatgctcttc tCCGCTGTGG CCCGAACGCC TCCCgctagc gatgggatct aatacgactc
     4381 actataggga gaccaaggga aaatagaatg aagtgatctc caaaaaataa gtactttttg
     4441 actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag
     4501 taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa
     4561 tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg ggatccagct
     4621 gcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     4681 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     4741 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     4801 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     4861 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     4921 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     4981 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     5041 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     5101 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     5161 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     5221 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     5281 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     5341 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     5401 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     5461 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcaaaaa
     5521 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5581 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5641 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5701 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5761 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5821 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5881 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5941 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6001 gatacctaca gcgtgagcta tgagaaagcg ccacggttcc cgaagggaga aaggcggaca
     6061 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6121 acgcctggta tctttatagt cctgt   
//
