LOCUS       sequence-242296-        4362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SaCas9 sgRNA expression vector for cloning own guides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4362)
  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 4362)
  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: SaCas9_sgRNA_expression_in_pBluescript
FEATURES             Location/Qualifiers
     source          1..4362
                     /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        1631..1706
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     primer_bind     complement(1734..1750)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1735..1751)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1775..1794)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1776..1794)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1804..1821)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1804..1820)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1813..1835)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1962..2417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2049..2068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2259..2280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2443..2547
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2548..2616,2617..3408)
                     /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(2766..2785)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3579..4167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4068..4087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4321..4338
                     /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 agctagcaga
     1621 acctgctgac gttttagtac tctggaaaca gaatctacta aaacaaggca aaatgccgtg
     1681 tttatctcgt caacttgttg gcgagatttt tttgaattcg atatcaagct tatcgatacc
     1741 gtcgacctcg agggggggcc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg
     1801 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     1861 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     1921 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     1981 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2041 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2101 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2161 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2221 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2281 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2341 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2401 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2461 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2521 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2581 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2641 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2701 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2761 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     2821 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2881 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2941 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3001 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3061 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3121 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     3181 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3241 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3301 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3361 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3421 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3481 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3541 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3601 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3661 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3721 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3781 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3841 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3901 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3961 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4021 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4081 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4141 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4201 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4261 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4321 agcgagtcag tgagcgagga agcggaagag cgcccaatac gc
//
