LOCUS       40924_17399        4236 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pEN-2xChimera, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4236)
  AUTHORS   Durr J, Papareddy R, Nakajima K, Gutierrez-Marcos J.
  TITLE     Highly efficient heritable targeted deletions of gene clusters and 
            non-coding regulatory regions in Arabidopsis using CRISPR/Cas9
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4236)
  AUTHORS   Durr J, Gutierrez-Marcos J.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-JAN-2017) School of Life Sciences, University of 
            Warwick, Gibbet Hill Campus, Coventry, West Midlands CV4 7AL, United
            Kingdom
REFERENCE   3  (bases 1 to 4236)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4236)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (19-JAN-2017) School of Life Sciences, University of Warwick, Gibbet
            Hill Campus, Coventry, West Midlands CV4 7AL, United Kingdom"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    52..151
                     /label=attL1
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      152..539
                     /label=U6-26
                     /note="U6-26"
                     /regulatory_class="promoter"
     misc_RNA        558..633
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(645..669)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      681..1068
                     /label=U6-26
                     /note="U6-26"
                     /regulatory_class="promoter"
     misc_feature    1085..1201
                     /label=sgRNA scaffold
                     /note="sgRNA scaffold"
     misc_RNA        1085..1160
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1172..1196)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1226..1325)
                     /label=attL2
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1361..1379)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1397..1413)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1421..1437)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(1445..1475)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1490..1511)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(1799..2387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2561..3418)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(3419..3523)
                     /label=AmpR promoter
     rep_origin      complement(3601..4056)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     4197..4213
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4220..4236
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg gcaaataatg
       61 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca atgctttttt
      121 ataatgccaa ctttgtacaa aaaagcaggc tctttttttc ttcttcttcg ttcatacagt
      181 ttttttttgt ttatcagctt acattttctt gaaccgtagc tttcgttttc ttctttttaa
      241 ctttccattc ggagtttttg tatcttgttt catagtttgt cccaggatta gaatgattag
      301 gcatcgaacc ttcaagaatt tgattgaata aaacatcttc attcttaaga tatgaagata
      361 atcttcaaaa ggcccctggg aatctgaaag aagagaagca ggcccattta tatgggaaag
      421 aacaatagta tttcttatat aggcccattt aagttgaaaa caatcttcaa aagtcccaca
      481 tcgcttagat aagaaaacga agctgagttt atatacagct agagtcgaag tagtgattgg
      541 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
      601 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagacccag ctttcttgta
      661 caaagttggc attactgcag ctttttttct tcttcttcgt tcatacagtt tttttttgtt
      721 tatcagctta cattttcttg aaccgtagct ttcgttttct tctttttaac tttccattcg
      781 gagtttttgt atcttgtttc atagtttgtc ccaggattag aatgattagg catcgaacct
      841 tcaagaattt gattgaataa aacatcttca ttcttaagat atgaagataa tcttcaaaag
      901 gcccctggga atctgaaaga agagaagcag gcccatttat atgggaaaga acaatagtat
      961 ttcttatata ggcccattta agttgaaaac aatcttcaaa agtcccacat cgcttagata
     1021 agaaaacgaa gctgagttta tatacagcta gagtcgaagt agtgattggg agacggacgt
     1081 ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     1141 aagtggcacc gagtcggtgc tttttttcta gacccagctt tcttgtacaa agttggcatt
     1201 aggatccgat atcaagcttc ccgggaccca gctttcttgt acaaagttgg cattataaaa
     1261 aataattgct catcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt
     1321 atttggagct cccaacgcgt tggatgcata gcttgagtat tctatagtgt cacctaaata
     1381 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1441 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1501 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1561 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1621 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1681 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1741 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1801 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1861 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1921 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1981 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2041 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2101 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2161 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2221 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2281 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2341 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2401 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2461 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2521 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2581 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2641 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2701 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2761 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2821 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     2881 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2941 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3001 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3061 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3121 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3181 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3241 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3301 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3361 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3421 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3481 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3541 tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     3601 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     3661 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     3721 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     3781 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     3841 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     3901 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     3961 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     4021 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
     4081 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     4141 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     4201 aacgacggcc agtgaattgt aatacgactc actata
//
