LOCUS       Exported                3348 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Golden Gate recipient and Gateway entry vector; assembly of 3 gRNAs 
            .
ACCESSION   .
VERSION     .
KEYWORDS    pYPQ143
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3348)
  AUTHORS   Lowder LG, Paul JW, Baltes NJ, Voytas DF, Zhang Y, Zhang D, Tang X, 
            Zheng X, Hsieh TF, Qi Y
  TITLE     A CRISPR/Cas9 toolbox for multiplexed plant genome editing and 
            transcriptional regulation.
  JOURNAL   Plant Physiol. 2015 Aug 21. pii: pp.00636.2015.
  PUBMED    26297141
REFERENCE   2  (bases 1 to 3348)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3348
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     202..218
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        228..246
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(294..314)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(294..312)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(333..349)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(346..368)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    357..373
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(381..411)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    426..447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(512..611)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(629..647)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(664..682)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(687..703)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1134..1925
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2018..2606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2507..2526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2760..2777
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      2936..2963
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      3055..3141
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3205..3221
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3237..3332
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3248..337)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGILRYLEGTPWGNFCIQSWHYKKALLINLLQRTGHYQSK"
ORIGIN
        1 ctagataacg caggatcccc aagtggtggc tatcgagacc ggcgccgcta cagggcgcgt
       61 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
      121 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      181 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
      241 tatagggcga attgggtacc gggccccccc tcgaggtcct ccagcttttg ttccctttag
      301 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      361 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      421 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccaccgg
      481 tggtctctcc agactagtaa gggcgaattc gacccagctt tcttgtacaa agttggcatt
      541 ataaaaaata attgctcatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
      601 atcattattt gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagca
      661 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
      721 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc ctcctctgga
      781 ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg tgacgcacac
      841 cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt
      901 aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg
      961 gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta
     1021 tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga
     1081 gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac atcatgaggg
     1141 aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc atcgagcgcc
     1201 atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga
     1261 agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc
     1321 ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc
     1381 tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag
     1441 ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca ggtatcttcg
     1501 agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg
     1561 ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat
     1621 ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggcgatg
     1681 agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg
     1741 cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag tatcagcccg
     1801 tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg
     1861 cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca
     1921 aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     1981 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2041 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2101 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2161 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2221 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2281 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2341 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2401 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2461 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2521 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2581 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2641 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2701 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2761 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     2821 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     2881 agcgcaacgc aattaatacg cgtaccgctc gccaggaaga gtttgtagaa acgcaaaaag
     2941 gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc
     3001 tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt
     3061 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga
     3121 ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta acgcttgcat
     3181 ggatgttttc ccagtcacga cgttgtaaaa cgacggccag tcttaagctc gggcccaaat
     3241 aatgatttta ttttgactga tagtgacctg ttcgttgcaa caaattgatg agcaatgctt
     3301 ttttataatg ccaactttgt atacaaaagt tgccccatgg cgttccct
//
