LOCUS       40924_45198        2845 bp DNA     circular SYN 13-MAY-2021
DEFINITION  gRNA expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2845)
  AUTHORS   Guo X, Zhang T, Hu Z, Zhang Y, Shi Z, Wang Q, Cui Y, Wang F, Zhao H,
            Chen Y
  TITLE     Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.
  JOURNAL   Development. 2014 Jan 8.
  PUBMED    24401372
REFERENCE   2  (bases 1 to 2845)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2845)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Development. 2014 Jan 8."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    55..76
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        91..121
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    129..145
                     /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     153..169
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        234..252
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(398..414)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(623..642)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     742..764
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(802..820)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        888..992
                     /label=AmpR promoter
     CDS             993..1850
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2024..2612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2766..2783
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
       61 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      121 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      181 caagctcgct tgcacgcctc tgcactcgtc ggtcccggca tccgataccc aattaatacg
      241 actcactata ggtcgtcttc gagaagacct gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt aaagacccaa
      361 tatctcgacg cattcgcgaa gtaccgatct ccaattcact ggccgtcgtt ttacaacgtc
      421 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
      481 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
      541 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
      601 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
      661 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
      721 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
      781 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
      841 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      901 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      961 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1021 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1081 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1141 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1201 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1261 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1321 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1381 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1441 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1501 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     1561 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1621 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1681 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1741 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1801 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1861 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1921 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1981 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2041 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2101 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2161 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2221 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2281 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2341 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2401 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2461 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2521 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2581 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2641 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2701 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2761 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     2821 ccgcgcgttg gccgattcat taatg
//
