LOCUS       sequence-122901-        3998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian U6-driven sgRNA (NMm1) targeting GTCCCCTCCACCCCACAGTG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3998)
  AUTHORS   Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM
  TITLE     Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.
  JOURNAL   Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681.
  PUBMED    24076762
REFERENCE   2  (bases 1 to 3998)
  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: "Nat 
            Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681."
COMMENT     Sequence Label: M-NM-sgRNA
FEATURES             Location/Qualifiers
     source          1..3998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        406..654
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     406..426
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     577..596
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        675..775
                     /label=Nm gRNA scaffold
                     /note="guide RNA scaffold for the Neisseria meningitidis 
                     CRISPR/Cas9 system (Esvelt et al., 2013)"
     protein_bind    complement(787..811)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(886..905)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(887..905)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(912..929)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(912..928)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(921..943)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1066..1368
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1247..1266
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1717..2511
                     /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             1717..2511
                     /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(1771..1790)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2381..2400
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2711..2720
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2717..3091
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      3229..3817
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3718..3737
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3971..3988
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gccctttgta caaaaaagca ggctttaaag
      361 gaaccaattc agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc
      421 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
      481 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      541 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
      601 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtcccc
      661 tccaccccac agtggttgta gctccctttc tcgaaagaga accgttgcta caataaggcc
      721 gtctgaaaag atgtgccgca acgctctgcc ccttaaagct tctgctttaa cgggcttttt
      781 ttctagaccc agctttcttg tacaaagttg gcattaaagg gcgaattctg cagatatcca
      841 tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg
      901 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
      961 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1021 cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac
     1081 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     1141 acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     1201 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     1261 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
     1321 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg
     1381 agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa
     1441 cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc
     1501 gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt
     1561 ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     1621 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     1681 agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     1741 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     1801 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     1861 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     1921 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     1981 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2041 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2101 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2161 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     2221 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     2281 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     2341 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     2401 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2461 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac
     2521 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2581 atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2641 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca
     2701 cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
     2761 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
     2821 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
     2881 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
     2941 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
     3001 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
     3061 cgtgcacttc gtggccgagg agcaggactg acacgtgcta aaacttcatt tttaatttaa
     3121 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3181 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3241 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3301 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3361 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3421 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3481 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3541 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3601 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3661 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3721 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3781 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3841 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3901 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3961 gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
