LOCUS       sequence-436605-        3963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA scaffold and synthetic U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3963)
  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 3963)
  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_sU6
FEATURES             Location/Qualifiers
     source          1..3963
                     /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"
     misc_RNA        345..420
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     511..531
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(851..870)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(852..870)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(877..894)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(877..893)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(886..908)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1031..1333
                     /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     1212..1231
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1682..2476
                     /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             1682..2476
                     /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(1736..1755)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2346..2365
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2676..2685
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2682..3056
                     /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      3194..3782
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3683..3702
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3936..3953
                     /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 gcccttgaag acctgtttta gagctagaaa
      361 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
      421 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
      481 gcgccaattc tgcagacaaa tggctctaga gagggcctat ttcccatgat tccttcatat
      541 ttgcatataa tatttcctag taactataga ggcttaatgt gcgataaaag acagataatc
      601 tgttcttttt aatactagct acattttaca tgataggctt ggatttctat aagagataca
      661 aatactaaat tattatttta aaaaacagca caaaaggaaa cttaccgtaa cttgaaagta
      721 attgtgtgtt ttgagacttt atatatgcat gcgagaaaag ccttgtttgg tttgggtctt
      781 caagggcgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg
      841 gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
      901 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
      961 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     1021 atacgtacgg cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt
     1081 ggatgtacag agtgatatta ttgacacgcc ggggcgacgg atggtgatcc ccctggccag
     1141 tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga
     1201 tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga
     1261 agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt
     1321 ctggggaata taaatgtcag gcatgagatt atcaaaaagg atcttcacct agatcctttt
     1381 cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc
     1441 tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac
     1501 atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg
     1561 ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc
     1621 aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag gatcgtttcg
     1681 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     1741 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     1801 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     1861 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     1921 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     1981 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     2041 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     2101 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     2161 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     2221 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     2281 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     2341 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     2401 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     2461 tgacgagttc ttctgaatta ttaacgctta caatttcctg atgcggtatt ttctccttac
     2521 gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa
     2581 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     2641 cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg
     2701 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     2761 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     2821 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     2881 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     2941 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     3001 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     3061 tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3121 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3181 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3241 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3301 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3361 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3421 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3481 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3541 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3601 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3661 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3721 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3781 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3841 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3901 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3961 aag
//
