LOCUS       40924_44958        3771 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Can use a PCR template to assemble new desired guide RNA. Contains 
            an Arabidopsis U6 promoter to drive guide RNA (targeting AtPDS3 gene
            target site 1) expression with a TTTTTT as terminator. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3771)
  AUTHORS   Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM,
            Sheen J
  TITLE     Multiplex and homologous recombination-mediated genome editing in 
            Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.
  JOURNAL   Nat Biotechnol. 2013 Aug;31(8):688-91. doi: 10.1038/nbt.2654.
  PUBMED    23929339
REFERENCE   2  (bases 1 to 3771)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3771)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2013-08"; volume: "31"; issue:
            "8"; pages: "688-91"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        407..710
                     /label=AtU6-1 promoter
                     /note="promoter for the Arabidopsis thaliana U6-1 snRNA
                     gene (Waibel and Filipowicz, 1990)"
     misc_RNA        731..806
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(818..842)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(900..916)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      1129..1584
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1601..1620)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     1720..1742
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1780..1798)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        1866..1970
                     /label=AmpR promoter
     CDS             1971..2828
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3002..3590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3744..3761
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgggcgc gccaagctta
      241 ggcctgtcga cgcccgggcg gtaccgcgat cgctcgcgac ctgcaggcat aaagccgtca
      301 gtgtccgcat aaagaaccac ccataatacc cataatagct gtttgccatc gctaccttag
      361 gaccgttata gttaattacc ctgttatccc tattaattaa gagctcagaa atctcaaaat
      421 tccggcagaa caattttgaa tctcgatccg tagaaacgag acggtcattg ttttagttcc
      481 accacgatta tatttgaaat ttacgtgagt gtgagtgaga cttgcataag aaaataaaat
      541 ctttagttgg gaaaaaattc aataatataa atgggcttga gaaggaagcg agggataggc
      601 ctttttctaa aataggccca tttaagctat taacaatctt caaaagtacc acagcgctta
      661 ggtaaagaaa gcagctgagt ttatatatgg ttagagacga agtagtgatt ggacttttgc
      721 cagccatggt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      781 ttgaaaaagt ggcaccgagt cggtgctttt tttctagacc cagctttctt gtacaaagtt
      841 ggcattaccc atcggcgcgc ctctagaatt taaatggatc ctacgtactc gaggaattca
      901 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
      961 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     1021 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
     1081 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     1141 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1201 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1261 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1321 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     1381 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1441 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     1501 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1561 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     1621 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     1681 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1741 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     1801 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     1861 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1921 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     1981 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2041 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2101 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2161 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     2221 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2281 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2341 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2401 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2461 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     2521 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     2581 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2641 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     2701 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     2761 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     2821 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     2881 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2941 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3001 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3061 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3121 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3661 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3721 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
