LOCUS       sequence-418948-        4026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA scaffold and mouse U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4026)
  AUTHORS   Vidigal JA, Ventura A
  TITLE     Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 
            libraries.
  JOURNAL   Nat Commun. 2015 Aug 17;6:8083. doi: 10.1038/ncomms9083.
  PUBMED    26278926
REFERENCE   2  (bases 1 to 4026)
  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 
            Commun."; date: "2015-08-17"; volume: "6:8083. doi"; pages: " 
            10.1038/ncomms9083"
COMMENT     Sequence Label: pDonor_mU6
FEATURES             Location/Qualifiers
     source          1..4026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    121..142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(157..162,163..180,181..187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    195..211
                     /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     200..222
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     219..235
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     219..235
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        253..271
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     253..270
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        359..434
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        531..844
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     763..783
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     complement(928..947)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(929..947)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(954..971)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(954..970)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(963..985)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1108..1410
                     /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     1289..1308
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1759..2553
                     /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             1759..2553
                     /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(1813..1832)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2423..2442
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2753..2762
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2759..3133
                     /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      3271..3859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3760..3779
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(4013..4026,1..4)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
       61 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      121 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      181 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      241 ttacgccaag ctatttaggt gacactatag aatactcaag ctatgcatca agcttggtac
      301 cgagctcgga tccactagta acggccgcca gtgtgctgga attcgccctt gaagacctgt
      361 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      421 caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc
      481 cgtttttagc gcgtgcgcca attctgcaga caaatggctc tagatctaga gatccgacgc
      541 cgccatctct aggcccgcgc cggccccctc gcacagactt gtgggagaag ctcggctact
      601 cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc ttaatgtgcg
      661 ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga taggcttgga
      721 tttctataag agatacaaat actaaattat tattttaaaa aacagcacaa aaggaaactc
      781 accctaactg taaagtaatt gtgtgttttg agactataaa tatgcatgcg agaaaagcct
      841 tgtttggttt gggtcttcaa gggcgaattc tgcagatatc catcacactg gcggccgctc
      901 gagcatgcat ctagagggcc caattcgccc tatagtgagt cgtattacaa ttcactggcc
      961 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     1021 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     1081 caacagttgc gcagcctata cgtacggcag tttaaggttt acacctataa aagagagagc
     1141 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgccggg gcgacggatg
     1201 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
     1261 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
     1321 gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
     1381 gccattaacc tgatgttctg gggaatataa atgtcaggca tgagattatc aaaaaggatc
     1441 ttcacctaga tccttttcac gtagaaagcc agtccgcaga aacggtgctg accccggatg
     1501 aatgtcagct actgggctat ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta
     1561 gcttgcagtg ggcttacatg gcgatagcta gactgggcgg ttttatggac agcaagcgaa
     1621 ccggaattgc cagctggggc gccctctggt aaggttggga agccctgcaa agtaaactgg
     1681 atggctttct tgccgccaag gatctgatgg cgcaggggat caagctctga tcaagagaca
     1741 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     1801 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     1861 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     1921 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     1981 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     2041 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     2101 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2161 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     2221 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     2281 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     2341 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     2401 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     2461 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     2521 gccttctatc gccttcttga cgagttcttc tgaattatta acgcttacaa tttcctgatg
     2581 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatcaggtg gcacttttcg
     2641 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2701 gctcatgaga caataaccct gataaatgct tcaataatag cacgtgagga gggccaccat
     2761 ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga
     2821 gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt
     2881 ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa
     2941 caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt
     3001 cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc
     3061 gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga
     3121 ggagcaggac tgacacgtgc taaaacttca tttttaattt aaaaggatct aggtgaagat
     3181 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3241 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3301 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3361 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3421 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3481 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3541 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3601 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3661 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3721 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3781 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3841 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3901 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3961 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4021 agtgag
//
