LOCUS       sequence-242295-        4356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SpCas9 sgRNA expression vector for cloning own guides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4356)
  AUTHORS   Bolukbasi MF, Liu P, Luk K, Kwok SF, Gupta A, Amrani N, Sontheimer 
            EJ, Zhu LJ, Wolfe SA
  TITLE     Orthogonal Cas9-Cas9 chimeras provide a versatile platform for 
            genome editing.
  JOURNAL   Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x.
  PUBMED    30451839
REFERENCE   2  (bases 1 to 4356)
  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: "2018-11-19"; volume: "9(1):4856. doi"; pages: " 
            10.1038/s41467-018-07310-x"
COMMENT     Sequence Label: SpCas9_sgRNA_expression_in_pBluescript
FEATURES             Location/Qualifiers
     source          1..4356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    93..114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(129..134,135..152,153..159)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    167..183
                     /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     172..194
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     191..207
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     191..207
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     226..246
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        228..246
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        293..541
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     293..313
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     464..483
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(672..1466)
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(783..802)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1393..1412
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        1625..1700
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1728..1744)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1729..1745)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1769..1788)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1770..1788)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1798..1815)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1798..1814)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1807..1829)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1956..2411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2043..2062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2253..2274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2437..2541
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2542..2610,2611..3402)
                     /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     complement(2760..2779)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3573..4161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4062..4081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4315..4332
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
       61 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
      121 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      181 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
      241 taaagggaac aaaagctgga gctccaccgc ggtggcggcc gcccccttca ccgagggcct
      301 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
      361 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
      421 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
      481 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
      541 cggtgtgcag gtgagtgatc caaacgcccg gcggcaaccg agcgttctga acaaatccag
      601 atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc tctcgaaccc
      661 cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
      721 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
      781 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
      841 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
      901 ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt
      961 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     1021 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     1081 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     1141 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     1201 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     1261 cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg
     1321 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
     1381 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
     1441 gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat
     1501 cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag
     1561 ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccgacggatc aagaacctgc
     1621 tgacgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     1681 aagtggcacc gagtcggtgc tttttttgaa ttcgatatca agcttatcga taccgtcgac
     1741 ctcgaggggg ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     1801 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     1861 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     1921 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     1981 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2041 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2101 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2161 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2221 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2281 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2341 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2401 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2461 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2521 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2581 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2641 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2701 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2761 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     2821 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2881 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2941 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3001 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3061 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3121 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3181 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3241 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3301 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3361 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3421 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3481 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3541 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3601 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3661 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3721 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3781 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3841 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3901 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3961 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4021 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4081 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4141 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4201 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4261 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4321 tcagtgagcg aggaagcgga agagcgccca atacgc
//
