LOCUS       sequence-266820-        3542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the S. pyogenes sgRNA from the mouse U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
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
REFERENCE   2  (bases 1 to 3542)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2014 Aug 13. pii: gku749."
COMMENT     Sequence Label: pmU6-gRNA
FEATURES             Location/Qualifiers
     source          1..3542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..314
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     233..253
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        332..407
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(452..472)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(452..470)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(491..507)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(491..507)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(504..526)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    515..531
                     /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(join(539..545,546..563,564..569))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    584..605
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(722..739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(893..1481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(973..992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1558..2352)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(1558..2352)
                     /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(1669..1688)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2279..2298
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2387..2744)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2401..2536
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2553..2744
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2701..2721
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(2746..2850)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2876..3331)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3013..3034)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3225..3244
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3458..3480
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3472..3489
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3473..3489
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3499..3518
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3499..3517
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gatccgacgc cgccatctct aggcccgcgc cggccccctc gcacagactt gtgggagaag
       61 ctcggctact cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc
      121 ttaatgtgcg ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga
      181 taggcttgga tttctataag agatacaaat actaaattat tattttaaaa aacagcacaa
      241 aaggaaactc accctaactg taaagtaatt gtgtgttttg agactataaa tatcccttgg
      301 agaaaagcct tgtttgtctt cgagaagacc tgttttagag ctagaaatag caagttaaaa
      361 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgtactg
      421 agacgccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
      481 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
      541 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
      601 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
      661 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
      721 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
      781 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
      841 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
      901 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      961 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1021 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1081 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1141 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1201 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1261 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1321 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1381 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1441 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1501 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgatca
     1561 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     1621 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     1681 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     1741 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
     1801 gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag
     1861 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     1921 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     1981 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga
     2041 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
     2101 gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
     2161 tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     2221 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     2281 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     2341 ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcgatctttg caaaagccta
     2401 ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggccgag gcggcctcgg
     2461 cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg
     2521 gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat
     2581 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg
     2641 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     2701 accctaactg acacacattc cacagctggt tctttccgcc tcaggactct tcctttttca
     2761 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2821 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     2881 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2941 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3001 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3061 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3121 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3181 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3241 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3301 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     3361 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     3421 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     3481 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac ccgtctcaga
     3541 ta
//
