LOCUS       sequence-322481-        3515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the S. pyogenes sgRNA from the human U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3515)
  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 3515)
  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: phU6-gRNA
FEATURES             Location/Qualifiers
     source          1..3515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..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        696..944
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     696..716
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     867..886
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        963..1038
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1094..1114)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1094..1112)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1133..1149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1133..1149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1146..1168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1157..1173
                     /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(1181..1187,1188..1205,1206..1211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1226..1247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1364..1381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1535..2123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1615..1634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2200..2994)
                     /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(2200..2994)
                     /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(2311..2330)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2921..2940
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3029..3386)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3043..3178
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3195..3386
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3343..3363
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3388..3492)
                     /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 tctcacaggc ggatcgatcc aaggtcgggc aggaagaggg cctatttccc atgattcctt
      721 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      781 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      841 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      901 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct tcgagaagac
      961 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     1021 gtggcaccga gtcggtgctt tttttctccg ctgagcgtac tgagacgccg cggtggagct
     1081 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     1141 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1201 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1321 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1381 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1441 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1501 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1561 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1621 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1681 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1741 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1801 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1861 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1921 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     1981 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2041 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2101 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2161 agtggaacga aaactcacgt taagggattt tggtcatgat cagaagaact cgtcaagaag
     2221 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     2281 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     2341 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     2401 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     2461 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     2521 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     2581 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     2641 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     2701 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     2761 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
     2821 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     2881 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     2941 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     3001 cgatcctcat cctgtctctt gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc
     3061 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa
     3121 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg
     3181 gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc
     3241 tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
     3301 tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat
     3361 tccacagctg gttctttccg cctcaggact cttccttttt caatattatt gaagcattta
     3421 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3481 aggggttccg cgcacatttc cccgaaaagt gccac
//
