LOCUS       ph7SK-gRNA                3504 bp ds-DNA     circular SYN 10-JAN-2018
DEFINITION  Expresses the S. pyogenes sgRNA from the 7SK promoter.
ACCESSION   .
VERSION     .
KEYWORDS    ph7SK-gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3504)
  AUTHORS   Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA
  TITLE     Multiplex CRISPR/Cas9-based genome engineering from a single 
            lentiviral vector.
  JOURNAL   Nucleic Acids Res. 2014 Aug 13. pii: gku749.
  PUBMED    25122746
COMMENT     This file is created by Vector NTI
            //gt12365.cn/
COMMENT     ORIGDB|GenBank
COMMENT     VNTAUTHORNAME|gt12365.cn|
COMMENT     VNTNAME|ph7SK-gRNA|
FEATURES             Location/Qualifiers
     source          1..3504
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        952..1027
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1083..1103)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1083..1101)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1122..1138)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1122..1138)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1135..1157)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1146..1162
                     /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(1170..1200)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1215..1236
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1353..1370)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1524..2112)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1604..1623)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2189..2983)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2300..2319)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2910..2929
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3018..3375)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3032..3167
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3086..3105)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3332..3352
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3377..3481)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacctt
      661 taattctagt actatgcatc gtctcattgt ctgcagtatt tagcatgccc cacccatctg
      721 caaggcattc tggatagtgt caaaacagcc ggaaatcaag tccgtttatc tcaaacttta
      781 gcattttggg aataaatgat atttgctatg ctggttaaat tagattttag ttaaatttcc
      841 tgctgaagct ctagtacgat aagcaacttg acctaagtgt aaagttgaga cttccttcag
      901 gtttatatag cttgtgcgcc gcttgggtac ctcgggtctt cgagaagacc tgttttagag
      961 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     1021 tcggtgcttt ttttctcgga ctgatgacct gagacgccgc ggtggagctc cagcttttgt
     1081 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     1141 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     1201 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     1261 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     1321 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1381 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1441 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1501 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1561 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1621 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1681 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1741 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1801 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1861 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1921 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     1981 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2041 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2101 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2161 aactcacgtt aagggatttt ggtcatgatc agaagaactc gtcaagaagg cgatagaagg
     2221 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt
     2281 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg
     2341 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat
     2401 tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct
     2461 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct
     2521 gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt
     2581 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga
     2641 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc
     2701 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa
     2761 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac
     2821 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
     2881 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
     2941 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
     3001 ctgtctcttg atcgatcttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc
     3061 tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag
     3121 ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg
     3181 ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     3241 gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt
     3301 ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccacagctgg
     3361 ttctttccgc ctcaggactc ttcctttttc aatattattg aagcatttat cagggttatt
     3421 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3481 gcacatttcc ccgaaaagtg ccac
//