LOCUS       pmU6-gRNA                3542 bp ds-DNA     circular SYN 10-JAN-2018
DEFINITION  Expresses the S. pyogenes sgRNA from the mouse U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pmU6-gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3542)
  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|pmU6-gRNA|
FEATURES             Location/Qualifiers
     source          1..3542
                     /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"
     promoter        670..983
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     902..922
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        1001..1076
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1121..1141)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1121..1139)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1160..1176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1160..1176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1173..1195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1184..1200
                     /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(1208..1238)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1253..1274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1391..1408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1562..2150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1642..1661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2227..3021)
                     /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(2338..2357)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2948..2967
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3056..3413)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3070..3205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3124..3143)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3370..3390
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3415..3519)
                     /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 tgggtacccg
      661 tctcagatag atccgacgcc gccatctcta ggcccgcgcc ggccccctcg cacagacttg
      721 tgggagaagc tcggctactc ccctgccccg gttaatttgc atataatatt tcctagtaac
      781 tatagaggct taatgtgcga taaaagacag ataatctgtt ctttttaata ctagctacat
      841 tttacatgat aggcttggat ttctataaga gatacaaata ctaaattatt attttaaaaa
      901 acagcacaaa aggaaactca ccctaactgt aaagtaattg tgtgttttga gactataaat
      961 atcccttgga gaaaagcctt gtttgtcttc gagaagacct gttttagagc tagaaatagc
     1021 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     1081 tttgtactga gacgccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg
     1141 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1201 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1261 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1321 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     1381 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1441 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1501 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1561 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1621 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     1681 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     1741 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1801 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1861 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     1921 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1981 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     2041 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2101 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2161 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2221 tcatgatcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc
     2281 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat
     2341 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc
     2401 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg
     2461 ggtcacgacg agatcctcgc cgtcgggcat gctcgccttg agcctggcga acagttcggc
     2521 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
     2581 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
     2641 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
     2701 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
     2761 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga
     2821 tagccgcgct gcctcgtctt gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa
     2881 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
     2941 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
     3001 caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cgatctttgc
     3061 aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg
     3121 cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg
     3181 aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact
     3241 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     3301 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     3361 actttccaca ccctaactga cacacattcc acagctggtt ctttccgcct caggactctt
     3421 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3481 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3541 ac
//