LOCUS       Exported                2936 bp ds-DNA     circular SYN 17-MAR-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2936)
  AUTHORS   Bikard Lab
  TITLE     Direct Submission
  JOURNAL   Exported Aug 29, 2017 from SnapGene 4.0.3
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..2936
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..18
                     /label=LC42
                     /note="ELZ2MutBB_R"
     primer_bind     73..92
                     /label=LC13
                     /note="pZS24-BB-R"
     primer_bind     complement(94..116)
                     /label=B377 pZ-F
                     /note="from pDB129 pZE21-CRISPR-bsaI"
     primer_bind     complement(103..125)
                     /label=B385
     CDS             209..1003
                     /codon_start=1
                     /label=kanR
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     misc_feature    1002..1031
                     /label=CBB CRISPR spapcer
                     /note="cut pELZ2mut1K backbone, cut pCRISPR, pZE21, pCRRNA 
                     backbone
                     "
     terminator      1034..1128
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1216..1804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1285..1304)
                     /label=LC79
                     /note="ccdBspa_R"
     primer_bind     complement(1833..1850)
                     /label=LC99
                     /note="OriAspa_R"
     primer_bind     1904..1923
                     /label=B248
                     /note="VR"
     primer_bind     1911..1971
                     /label=LC75_CRRNAcos_R
     misc_feature    1951..2174
                     /label=Minimal lambda cos sequence
     primer_bind     1951..1971
                     /label=LC91
                     /note="Cas9cos_R"
     primer_bind     2074..2089
                     /label=B160
                     /note="rpsLsp-R"
     primer_bind     complement(2084..2099)
                     /label=LC118
                     /note="treF_F"
     misc_feature    2086..2097
                     /label=Lambda cos site
     primer_bind     complement(2153..2214)
                     /label=LC74_CRRNAcos_F
     primer_bind     complement(2153..2174)
                     /label=LC90
                     /note="Cas9cos_F"
     terminator      2189..2275
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2272..2291
                     /label=LC20
                     /note="Ku-Seq7-R"
     primer_bind     complement(2287..2305)
                     /label=LC41
                     /note="ELZ2mutBB_F"
     primer_bind     complement(2288..2304)
                     /label=LC11
                     /note="pZS24-BB-F"
     primer_bind     2289..2313
                     /label=LC102
                     /note="pITCRRNA_F"
     primer_bind     2299..2329
                     /label=B298 - BamHI-leader-F
                     /note="from pACYC184"
     misc_feature    2305..2436
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     CDS             2305..2334
                     /codon_start=2
                     /locus_tag="SPy_1049"
                     /old_locus_tag="SPy1049"
                     /product="hypothetical protein"
                     /label='SPy_1049
                     /note="Best Blastp hit = pir||T03910 
                     UDP-glucuronosyltransferase homolog F39G3.1 - 
                     Caenorhabditis elegans >gi|2291205|gb|AAB65327.1| 
                     (AF016424) Similar to UDP-glucosyltransferase 
                     [Caenorhabditis elegans]"
                     /note="hypothetical protein"
                     /db_xref="GI:13622196"
                     /protein_id="AAK33939.1"
                     /translation="IS**LKIWY"
     primer_bind     2358..2377
                     /label=D016
                     /note="pDB114 RSR F"
     primer_bind     complement(2414..2436)
                     /label=B220
                     /note="leader-sp1-R"
     primer_bind     2417..2467
                     /label=B690
                     /note="SpCRISPR-F"
     primer_bind     2426..2446
                     /label=B361
                     /note="(pTrc)-crispr-F"
     primer_bind     complement(2428..2538)
                     /label=D020
                     /note="Spa 2 R"
     primer_bind     2437..2532
                     /label=D003
                     /note="Spacer spa 1"
     primer_bind     2437..2532
                     /label=D004
                     /note="Spacer spa 2"
     repeat_region   2437..2472
                     /label=CRISPR repeat
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     primer_bind     2437..2465
                     /label=D037
     primer_bind     2437..2461
                     /label=B259
                     /note="bgaA-sp1-F"
     primer_bind     2437..2459
                     /label=D035
                     /note="phiNM4 F"
     primer_bind     2437..2459
                     /label=B217
                     /note="srtA-sp1-F"
     primer_bind     2437..2459
                     /label=B219
                     /note="sp1-rep-F"
     primer_bind     2437..2459
                     /label=B684
                     /note="(protAsp)rep-F"
     primer_bind     2437..2459
                     /label=D025
                     /note="GyrA F pDB269"
     primer_bind     2437..2459
                     /label=D029
                     /note="hla 1 F pDB269"
     primer_bind     2437..2459
                     /label=D031
                     /note="hla 2 F pDB269"
     primer_bind     2437..2459
                     /label=B223
                     /note="Pyo-BsaIsp-F"
     primer_bind     2437..2458
                     /label=B321
                     /note="bsaI-sp-F"
     primer_bind     complement(2443..2472)
                     /label=B691
                     /note="SpCRISPR-R"
     primer_bind     complement(2444..2525)
                     /label=D038
                     /note="GG hla 2 R"
     primer_bind     complement(2448..2473)
                     /label=B260
                     /note="bgaA-sp1-R"
     primer_bind     complement(2449..2472)
                     /label=B224
                     /note="Pyo-BsaIsp-R"
     primer_bind     complement(2450..2473)
                     /label=D032
                     /note="hla 2 R pDB269"
     primer_bind     complement(2450..2473)
                     /label=D026
                     /note="GyrA R pDB269"
     primer_bind     complement(2450..2472)
                     /label=D030
                     /note="hla 1 R pDB269"
     primer_bind     complement(2450..2472)
                     /label=B685
                     /note="(protAsp)rep-R"
     primer_bind     complement(2450..2472)
                     /label=B218
                     /note="srtA-sp1-R"
     primer_bind     complement(2450..2472)
                     /label=D036
                     /note="phiNM4 R"
     primer_bind     2451..2531
                     /label=D037
     primer_bind     complement(2451..2472)
                     /label=D007
                     /note="spacer R"
     primer_bind     complement(2451..2472)
                     /label=B320
                     /note="bsaI-sp-R"
     primer_bind     complement(2456..2474)
                     /label=LC26
                     /note="ELZ1n2-R2"
     primer_bind     complement(2456..2473)
                     /label=B639
                     /note="mecA_dbsp3-R"
     primer_bind     complement(2456..2472)
                     /label=B657
                     /note="pUSA2_2sp"
     primer_bind     complement(2456..2472)
                     /label=B641
                     /note="sek_dbsp-R"
     misc_feature    2473..2502
                     /label=GFP 0 mismatch
     primer_bind     2502..2533
                     /label=B690
                     /note="SpCRISPR-F"
     primer_bind     2503..2614
                     /label=D018
                     /note="Spa 1 F"
     primer_bind     2503..2614
                     /label=D019
                     /note="Spa 2 F"
     primer_bind     2503..2614
                     /label=D021
                     /note="arlR F"
     primer_bind     2503..2614
                     /label=D022
                     /note="arlS F"
     primer_bind     2503..2598
                     /label=D003
                     /note="Spacer spa 1"
     primer_bind     2503..2598
                     /label=D004
                     /note="Spacer spa 2"
     repeat_region   2503..2538
                     /label=CRISPR repeat
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     primer_bind     2503..2527
                     /label=B259
                     /note="bgaA-sp1-F"
     primer_bind     2503..2525
                     /label=D035
                     /note="phiNM4 F"
     primer_bind     2503..2525
                     /label=B217
                     /note="srtA-sp1-F"
     primer_bind     2503..2525
                     /label=B219
                     /note="sp1-rep-F"
     primer_bind     2503..2525
                     /label=B684
                     /note="(protAsp)rep-F"
     primer_bind     2503..2525
                     /label=D025
                     /note="GyrA F pDB269"
     primer_bind     2503..2525
                     /label=D029
                     /note="hla 1 F pDB269"
     primer_bind     2503..2525
                     /label=D031
                     /note="hla 2 F pDB269"
     primer_bind     2503..2525
                     /label=B223
                     /note="Pyo-BsaIsp-F"
     primer_bind     2503..2524
                     /label=B321
                     /note="bsaI-sp-F"
     primer_bind     complement(2509..2538)
                     /label=B691
                     /note="SpCRISPR-R"
     primer_bind     complement(2510..2591)
                     /label=D038
                     /note="GG hla 2 R"
     primer_bind     complement(2514..2538)
                     /label=B260
                     /note="bgaA-sp1-R"
     primer_bind     complement(2515..2540)
                     /label=B224
                     /note="Pyo-BsaIsp-R"
     primer_bind     complement(2516..2541)
                     /label=D030
                     /note="hla 1 R pDB269"
     primer_bind     complement(2516..2539)
                     /label=D036
                     /note="phiNM4 R"
     primer_bind     complement(2516..2538)
                     /label=D032
                     /note="hla 2 R pDB269"
     primer_bind     complement(2516..2538)
                     /label=D026
                     /note="GyrA R pDB269"
     primer_bind     complement(2516..2538)
                     /label=B685
                     /note="(protAsp)rep-R"
     primer_bind     complement(2516..2538)
                     /label=B218
                     /note="srtA-sp1-R"
     primer_bind     2517..2597
                     /label=D037
     primer_bind     complement(2517..2538)
                     /label=D007
                     /note="spacer R"
     primer_bind     complement(2517..2538)
                     /label=B320
                     /note="bsaI-sp-R"
     primer_bind     complement(2522..2540)
                     /label=B657
                     /note="pUSA2_2sp"
     primer_bind     complement(2522..2538)
                     /label=LC26
                     /note="ELZ1n2-R2"
     primer_bind     complement(2522..2538)
                     /label=B641
                     /note="sek_dbsp-R"
     primer_bind     complement(2522..2538)
                     /label=B639
                     /note="mecA_dbsp3-R"
     misc_feature    2539..2568
                     /label=mCherry 0 mismatches
     primer_bind     2568..2591
                     /label=D031
                     /note="hla 2 F pDB269"
     primer_bind     2568..2591
                     /label=B223
                     /note="Pyo-BsaIsp-F"
     repeat_region   2569..2604
                     /label=CRISPR repeat
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     primer_bind     2569..2599
                     /label=B690
                     /note="SpCRISPR-F"
     primer_bind     2569..2593
                     /label=B259
                     /note="bgaA-sp1-F"
     primer_bind     2569..2591
                     /label=D035
                     /note="phiNM4 F"
     primer_bind     2569..2591
                     /label=B217
                     /note="srtA-sp1-F"
     primer_bind     2569..2591
                     /label=B219
                     /note="sp1-rep-F"
     primer_bind     2569..2591
                     /label=B684
                     /note="(protAsp)rep-F"
     primer_bind     2569..2591
                     /label=D025
                     /note="GyrA F pDB269"
     primer_bind     2569..2591
                     /label=D029
                     /note="hla 1 F pDB269"
     primer_bind     2569..2590
                     /label=B321
                     /note="bsaI-sp-F"
     primer_bind     complement(2575..2625)
                     /label=B691
                     /note="SpCRISPR-R"
     primer_bind     complement(2576..2604)
                     /label=D038
                     /note="GG hla 2 R"
     primer_bind     2580..2620
                     /label=B374
                     /note="rep_mut-F"
     primer_bind     complement(2580..2604)
                     /label=B260
                     /note="bgaA-sp1-R"
     primer_bind     complement(2581..2604)
                     /label=B224
                     /note="Pyo-BsaIsp-R"
     primer_bind     complement(2582..2605)
                     /label=B218
                     /note="srtA-sp1-R"
     primer_bind     complement(2582..2604)
                     /label=D032
                     /note="hla 2 R pDB269"
     primer_bind     complement(2582..2604)
                     /label=D030
                     /note="hla 1 R pDB269"
     primer_bind     complement(2582..2604)
                     /label=D026
                     /note="GyrA R pDB269"
     primer_bind     complement(2582..2604)
                     /label=B685
                     /note="(protAsp)rep-R"
     primer_bind     complement(2582..2604)
                     /label=D036
                     /note="phiNM4 R"
     primer_bind     complement(2583..2605)
                     /label=B320
                     /note="bsaI-sp-R"
     primer_bind     complement(2583..2604)
                     /label=D007
                     /note="spacer R"
     primer_bind     complement(2588..2604)
                     /label=LC26
                     /note="ELZ1n2-R2"
     primer_bind     complement(2588..2604)
                     /label=B657
                     /note="pUSA2_2sp"
     primer_bind     complement(2588..2604)
                     /label=B641
                     /note="sek_dbsp-R"
     primer_bind     complement(2588..2604)
                     /label=B639
                     /note="mecA_dbsp3-R"
     primer_bind     2604..2626
                     /label=Tracr F
     primer_bind     2605..2667
                     /label=LC35_tracrRNA_R
     misc_feature    2605..2639
                     /label=pre-tracr sequence
     primer_bind     complement(2607..2639)
                     /label=B299 - EcoRI-rep-R
                     /note="from pACYC184"
     primer_bind     complement(2607..2630)
                     /label=B527
                     /note="SphI-CRISPR-R"
     primer_bind     complement(2607..2630)
                     /label=B333
                     /note="(cat)-CRISPR-R"
     primer_bind     complement(2607..2630)
                     /label=B328
                     /note="(pC194)-cas9-R"
     misc_feature    2640..2810
                     /label=tracrRNA
                     /note="tracrRNA"
                     /note="tracrRNA"
     primer_bind     2723..2744
                     /label=B260
                     /note="bgaA-sp1-R"
     primer_bind     2723..2743
                     /label=B224
                     /note="Pyo-BsaIsp-R"
     primer_bind     2723..2742
                     /label=D032
                     /note="hla 2 R pDB269"
     primer_bind     2723..2742
                     /label=D030
                     /note="hla 1 R pDB269"
     primer_bind     2723..2742
                     /label=D026
                     /note="GyrA R pDB269"
     primer_bind     2723..2742
                     /label=B685
                     /note="(protAsp)rep-R"
     primer_bind     2723..2742
                     /label=B218
                     /note="srtA-sp1-R"
     primer_bind     2723..2742
                     /label=D036
                     /note="phiNM4 R"
     primer_bind     2723..2741
                     /label=D007
                     /note="spacer R"
     primer_bind     2723..2741
                     /label=B320
                     /note="bsaI-sp-R"
     primer_bind     complement(2731..2747)
                     /label=B259
                     /note="bgaA-sp1-F"
     primer_bind     complement(2733..2747)
                     /label=D035
                     /note="phiNM4 F"
     primer_bind     complement(2733..2747)
                     /label=B217
                     /note="srtA-sp1-F"
     primer_bind     complement(2733..2747)
                     /label=B219
                     /note="sp1-rep-F"
     primer_bind     complement(2733..2747)
                     /label=B684
                     /note="(protAsp)rep-F"
     primer_bind     complement(2733..2747)
                     /label=D025
                     /note="GyrA F pDB269"
     primer_bind     complement(2733..2747)
                     /label=D029
                     /note="hla 1 F pDB269"
     primer_bind     complement(2733..2747)
                     /label=D031
                     /note="hla 2 F pDB269"
     primer_bind     complement(2733..2747)
                     /label=B223
                     /note="Pyo-BsaIsp-F"
     primer_bind     complement(2734..2747)
                     /label=B321
                     /note="bsaI-sp-F"
     misc_feature    2811..2840
                     /label=post-tracr sequence
     primer_bind     complement(2818..2842)
                     /label=dCas9-tracr R
     primer_bind     complement(2818..2840)
                     /label=Tracr R
     primer_bind     2912..2934
                     /label=B254
                     /note="tracrRNA-R"
     primer_bind     complement(2916..2935)
                     /label=LC103
                     /note="pITCRRNA_R"
     primer_bind     complement(2919..22)
                     /label=LC36_tracrRNA_F
     primer_bind     2919..22
                     /label=LC34_pCRISPR_BB_R
ORIGIN
        1 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
       61 ttcttagacg tcggaattgc cagctggggc gccctctggt aaggttggga agccctgcaa
      121 agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat caagatctga
      181 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
      241 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
      301 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
      361 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
      421 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
      481 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
      541 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
      601 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
      661 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
      721 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
      781 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
      841 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
      901 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
      961 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     1021 gagagctcgc ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat
     1081 ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aagcactagt
     1141 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1201 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1261 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1321 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1381 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1441 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1501 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1561 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagcatt gagaaagcgc
     1621 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1681 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1741 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1801 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1861 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1921 agctgatacc gctcgccgca gccgaacgcc ccaaaaagcc tcgctttcag cacctgtcgt
     1981 ttcctttctt ttcagagggt attttaaata aaaacattaa gttatgacga agaagaacgg
     2041 aaacgcctta aaccggaaaa ttttcataaa tagcgaaaac ccgcgaggtc gccgccccgt
     2101 aacctgtcgg atcaccggaa aggacccgta aagtgataat gattatcatc tacatatcac
     2161 aacgtgcgtg gagggtctag ggcggcggat ttgtcctact caggagagcg ttcaccgaca
     2221 aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc
     2281 tctagcacgc gtaccatggg atcctatttc ttaataacta aaaatatggt ataatactct
     2341 taataaatgc agtaatacag gggcttttca agactgaagt ctagctgaga caaatagtgc
     2401 gattacgaaa ttttttagac aaaaatagtc tacgaggttt tagagctatg ctgttttgaa
     2461 tggtcccaaa acccttcacc ttcaccacga acagagaatt tggttttaga gctatgctgt
     2521 tttgaatggt cccaaaacag tttggcggtc tgggtgcctt catacggagt tttagagcta
     2581 tgctgttttg aatggtccca aaacttcagc acactgagac ttgttgagtt gaattcatga
     2641 gtattaagta ttgttttatg gctgataaat ttctttgaat ttctccttga ttatttgtta
     2701 taaaagttat aaaataatct tgttggaacc attcaaaaca gcatagcaag ttaaaataag
     2761 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gatacttcta
     2821 ttctactctg actgcaaacc aaaaaaacaa gcgctttcaa aacgcttgtt ttatcatttt
     2881 tagggaaatt aatctcttaa tccttttatc attctacatt taggcgctgc catctt
//
