LOCUS       sequence-232787-        4356 bp    DNA     circular SYN 14-APR-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 Apr 14, 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"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2236..2484
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2236..2256
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2407..2426
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(2615..3409)
                     /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(2726..2745)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3336..3355
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        3568..3643
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(3671..3687)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3672..3688)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3712..3731)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3713..3731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3741..3758)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3741..3757)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3750..3772)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3899..4354
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3986..4005)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4196..4217
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg
     2221 gccgccccct tcaccgaggg cctatttccc atgattcctt catatttgca tatacgatac
     2281 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     2341 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     2401 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     2461 atcttgtgga aaggacgaaa caccggtgtg caggtgagtg atccaaacgc ccggcggcaa
     2521 ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg
     2581 agtccaagcg agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat
     2641 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     2701 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     2761 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     2821 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     2881 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
     2941 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
     3001 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     3061 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     3121 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     3181 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca
     3241 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     3301 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     3361 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     3421 ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga
     3481 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     3541 attccgacgg atcaagaacc tgctgacgtt ttagagctag aaatagcaag ttaaaataag
     3601 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaattcgata
     3661 tcaagcttat cgataccgtc gacctcgagg gggggcccgg tacccaattc gccctatagt
     3721 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3781 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     3841 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     3901 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3961 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4021 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4081 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4141 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4201 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4261 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4321 gcgaatttta acaaaatatt aacgcttaca atttag
//
