LOCUS       40924_42334        2541 bp DNA     circular SYN 13-MAY-2021
DEFINITION  gRNA cloning vector for in vitro transcription of target gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2541)
  AUTHORS   Jao LE, Wente SR, Chen W
  TITLE     Efficient multiplex biallelic zebrafish genome editing using a 
            CRISPR nuclease system.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Aug 5.
  PUBMED    23918387
REFERENCE   2  (bases 1 to 2541)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2541)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl
            Acad Sci U S A. 2013 Aug 5."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     195..211
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(239..314)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(348..366)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(390..406)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(414..430)
                     /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(438..468)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(483..504)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(621..638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(792..1380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1554..2411)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2412..2516)
                     /label=AmpR promoter
ORIGIN
        1 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
       61 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
      121 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
      181 ccagtcacga cgttgtaaaa cgacggccag tgaattcgag ctcggtaccc ggggatccgc
      241 accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta acttgctatt
      301 tctagctcta aaacagagac ggtcgactca gactagatct cgtctcccct atagtgagtc
      361 gtattaattt caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
      421 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
      481 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
      541 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
      601 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
      661 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
      721 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      781 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      841 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      901 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
      961 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     1021 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1081 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1141 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1201 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     1261 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1321 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1381 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1441 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1501 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     1561 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     1621 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     1681 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     1741 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     1801 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     1861 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     1921 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     1981 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2041 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     2101 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     2161 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     2221 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2281 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2341 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2401 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     2461 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     2521 tttccccgaa aagtgccacc t
//
