LOCUS       sequence-99803-e        3270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty backbone for cloning N. meningiditis protospacers. Encodes 
            constant region of N. meningiditis Cas9 synthetic gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3270)
  AUTHORS   Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy
            TE, Gersbach CA
  TITLE     Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates
            genes from promoters and enhancers.
  JOURNAL   Nat Biotechnol. 2015 May;33(5):510-7. doi: 10.1038/nbt.3199. Epub 
            2015 Apr 6.
  PUBMED    25849900
REFERENCE   2  (bases 1 to 3270)
  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"; doi: "10.1038/nbt.3199"; 
            journalName: "Nat Biotechnol"; date: "2015-05"; volume: "33"; issue:
            "5"; pages: "510-7"
COMMENT     Sequence Label: pZDonor-Nm-Cas9-gRNA-hU6
FEATURES             Location/Qualifiers
     source          1..3270
                     /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        661..909
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     832..851
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1082..1102)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1082..1100)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1121..1137)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1121..1137)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1134..1156)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1145..1161
                     /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(1169..1175,1176..1193,1194..1199))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1214..1235
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1352..1369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1523..2111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1603..1622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2282..3073,3074..3142))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2905..2924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3143..3247)
                     /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 tgggtaccaa
      661 gcttgcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      721 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      781 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      841 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      901 cgaaacaccg ggtcttcgag aagacctgtt gtagctccct ttctcatttc ggaaacgaaa
      961 tgagaaccgt tgctacaata aggccgtctg aaaagatgtg ccgcaacgct ctgcccctta
     1021 aagcttctgc tttaaggggc tttttttgcg gccgcccgcg gtggagctcc agcttttgtt
     1081 ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     1141 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     1201 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     1261 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1321 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     1381 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     1441 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     1501 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     1561 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     1621 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     1681 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     1741 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     1801 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     1861 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     1921 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     1981 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2041 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2101 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2161 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     2221 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     2281 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     2341 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     2401 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     2461 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     2521 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     2581 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     2641 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     2701 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     2761 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     2821 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     2881 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     2941 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3001 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     3061 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     3121 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     3181 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3241 ttccgcgcac atttccccga aaagtgccac
//
