LOCUS       sequence-431371-        6422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid encodes sgRNA that target Renilla luciferase with 
            stretch sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6422)
  AUTHORS   Ui-Tei K, Maruyama S, Nakano Y
  TITLE     Enhancement of single guide RNA transcription for efficient 
            CRISPR/Cas-based genomic engineering.
  JOURNAL   Genome. 2017 Jun;60(6):537-545. doi: 10.1139/gen-2016-0127. Epub 
            2017 Jan 26.
  PUBMED    28177825
REFERENCE   2  (bases 1 to 6422)
  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: 
            "10.1139/gen-2016-0127"; journalName: "Genome"; date: "2017-06"; 
            volume: "60"; issue: "6"; pages: "537-545"
COMMENT     Sequence Label: pSuperCRISPR-sgRNA-RL69-ATTT
FEATURES             Location/Qualifiers
     source          1..6422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     1421..1437
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1422..1438
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(1453..1528)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1557..1771)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(1617..1636)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        1782..2281
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1817..1840)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2214..2233
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2306..2905
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2306..2325)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2802..2822
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    3491..3507
                     /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(3515..3521,3522..3539,3540..3545))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3560..3581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3698..3715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3869..4457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3949..3968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4628..5419,5420..5488))
                     /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     5251..5270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5489..5593)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5661..5679
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5717..5739)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5839..5858
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6052..6074
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6202..6221
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6421..6422,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatcgatctc tcgaggtcga cggtatcgat
     1441 aagcttaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttaa
     1501 tttaacttgc tatttctagc tctaaatcgc acgttcattt gcttgcagcg ggatctgtgg
     1561 tctcatacag aacttataag attcccaaat ccaaagacat ttcacgttta tggtgatttc
     1621 ccagaacaca tagcgacatg caaatattgc agggcgccac tcccctgtcc ctcacagcca
     1681 tcttcctgcc agggcgcacg cgcgctgggt gttcccgcct agtgacactg ggcccgcgat
     1741 tccttggagc gggttgatga cgtcagcgtt cgaattctac cgggtagggg aggcgctttt
     1801 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     1861 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     1921 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     1981 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     2041 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     2101 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     2161 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     2221 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
     2281 gacctgcagc ccaagctagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac
     2341 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     2401 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     2461 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     2521 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     2581 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     2641 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     2701 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     2761 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     2821 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     2881 catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag
     2941 gagcgcacga ccccatgcat cgataaaata aaagatttta tttagtctcc agaaaaaggg
     3001 gggaatgaaa gaccccacct gtaggtttgg caagctagag aaccatcaga tgtttccagg
     3061 gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc
     3121 tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac
     3181 tcggcgcgcc agtcctccga tagactgcgt cgcccgggta cccgtgtatc caataaaccc
     3241 tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga
     3301 ttgactaccc gtcagcgggg gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac
     3361 attctgaggg taggagtcga atattaagta atcctgactc aattagccac tgttttgaat
     3421 ccacatactc caatactcct gaaatagttc attatggaca gcgcagaaag agctggggag
     3481 aattgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3541 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3601 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3661 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3721 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3781 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3841 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3901 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3961 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4021 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4081 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4141 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4201 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4261 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4321 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4381 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4441 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4501 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4561 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4621 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4681 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4741 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4801 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4861 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4921 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4981 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5041 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5101 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5161 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5221 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5281 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5341 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5401 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5461 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5521 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5581 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     5641 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     5701 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     5761 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     5821 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     5881 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
     5941 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     6001 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     6061 gacgttgtaa aacgacggcg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     6121 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     6181 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     6241 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg cgattagtcc aatttgttaa
     6301 agacaggata tcagtggtcc aggctctagt tttgactcaa caatatcacc agctgaagcc
     6361 tatagagtac gagccataga taaaataaaa gattttattt agtctccaga aaaagggggg
     6421 aa
//
