LOCUS       sequence-450757-        3261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-sgRNA (recipient). Expresses U6-sgRNA (recipient) for VLP 
            production. This is a recipient vector for cloning of specific 
            SpyCas9 sgRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3261)
  AUTHORS   Ramadoss GN, Namaganda SJ, Kumar MM, Hamilton JR, Sharma R, Chow KG,
            Workley LA, Macklin BL, Sun M, Ha AS, Liu JC, Fellmann C, Watry HL, 
            Dierks PH, Bose RS, Jin J, Perez BS, Sandoval Espinoza CR, Matia MP,
            Lu SH, Judge LM, Shy BR, Nussenzweig A, Adamson B, Murthy N, Doudna 
            JA, Kampmann M, Conklin BR
  TITLE     Characterizing and controlling CRISPR repair outcomes in nondividing
            human cells.
  JOURNAL   Nat Commun. 2025 Nov 17;16(1):9883. doi: 10.1038/s41467-025-66058-3.
  PUBMED    41249169
REFERENCE   2  (bases 1 to 3261)
  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: "2025-11-17"; volume: "16(1):9883. doi"; pages: " 
            10.1038/s41467-025-66058-3"
COMMENT     Sequence Label: pCF142_U6-sgRNA
FEATURES             Location/Qualifiers
     source          1..3261
                     /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     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        680..928
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     680..700
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     851..870
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        complement(1073..1091)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1112..1128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1112..1128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1125..1147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1136..1152
                     /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(1160..1166,1167..1184,1185..1190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1205..1226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1343..1360)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1514..2102)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1594..1613)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2273..3064,3065..3133))
                     /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     2896..2915
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3134..3238)
                     /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 gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctccaccg
      661 cggtggcggc cgctctagag agggcctatt tcccatgatt ccttcatatt tgcatatacg
      721 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
      781 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      841 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      901 atatatcttg tggaaaggac gaaacaccgg agacggagga cgacgaacgt ctctgtttaa
      961 gagctatgct ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa
     1021 aagtggcacc gagtcggtgc tttttttgaa ttctaggtac cagcttttgt tccctttagt
     1081 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     1141 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     1201 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1261 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1321 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1381 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1441 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1501 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1561 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1621 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1681 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1741 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1801 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1861 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1921 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     1981 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2041 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2101 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2161 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2221 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2281 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2341 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2401 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     2461 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     2521 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     2581 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     2641 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     2701 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     2761 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     2821 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     2881 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     2941 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3001 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3061 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3121 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3181 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3241 catttccccg aaaagtgcca c
//
