LOCUS       sequence-431350-        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-TTAT
FEATURES             Location/Qualifiers
     source          1..6422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    565..906
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             973..1389
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1319..1341
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1357..1373
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     1407..1423
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1408..1424
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(1439..1514)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1543..1757)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(1603..1622)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        1768..2267
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1803..1826)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2200..2219
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2292..2891
                     /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(2292..2311)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2788..2808
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    3477..3493
                     /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(3501..3507,3508..3525,3526..3531))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3546..3567
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3684..3701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3855..4443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3935..3954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4614..5405,5406..5474))
                     /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     5237..5256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5475..5579)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5647..5665
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5703..5725)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5825..5844
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6038..6060
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6188..6207
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6407..6422,1..501)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aatacataac
       61 tgagaataga gaagttcaga tcaaggttag gaacagagag acagcagaat atgggccaaa
      121 caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca
      181 gatgcggtcc cgccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa
      241 ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg
      301 ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca ctcggcgcgc
      361 cagtcctccg atagactgcg tcgcccgggt acccgtattc ccaataaagc ctcttgctgt
      421 ttgcatccga atcgtggact cgctgatcct tgggagggtc tcctcagatt gattgactgc
      481 ccacctcggg ggtctttcat ttggaggttc caccgagatt tggagacccc tgcccaggga
      541 ccaccgaccc ccccgccggg aggtaagctg gccagcggtc gtttcgtgtc tgtctctgtc
      601 tttgtgcgtg tttgtgccgg catctaatgt ttgcgcctgc gtctgtacta gttagctaac
      661 tagctctgta tctggcggac ccgtggtgga actgacgagt tctgaacacc cggccgcaac
      721 cctgggagac gtcccaggga ctttgggggc cgtttttgtg gcccgacctg aggaagggag
      781 tcgatgtgga atccgacccc gtcaggatat gtggttctgg taggagacga gaacctaaaa
      841 cagttcccgc ctccgtctga atttttgctt tcggtttgga accgaagccg cgcgtcttgt
      901 ctgctgcagc gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg
      961 tctgaaaatt agggccagac tgttaccact cccttaagtt tgaccttagg tcactggaaa
     1021 gatgtcgagc ggatcgctca caaccagtcg gtagatgtca agaagagacg ttgggttacc
     1081 ttctgctctg cagaatggcc aacctttaac gtcggatggc cgcgagacgg cacctttaac
     1141 cgagacctca tcacccaggt taagatcaag gtcttttcac ctggcccgca tggacaccca
     1201 gaccaggtcc cctacatcgt gacctgggaa gccttggctt ttgacccccc tccctgggtc
     1261 aagccctttg tacaccctaa gcctccgcct cctcttcctc catccgcccc gtctctcccc
     1321 cttgaacctc ctcgttcgac cccgcctcga tcctcccttt atccagccct cactccttct
     1381 ctaggcgccg gaattagatc gatctctcga ggtcgacggt atcgataagc ttaaaaaagc
     1441 accgactcgg tgccactttt tcaagttgat aacggactag ccttattata acttgctatt
     1501 tctagctcta taacgcacgt tcatttgctt gcagcgggat ctgtggtctc atacagaact
     1561 tataagattc ccaaatccaa agacatttca cgtttatggt gatttcccag aacacatagc
     1621 gacatgcaaa tattgcaggg cgccactccc ctgtccctca cagccatctt cctgccaggg
     1681 cgcacgcgcg ctgggtgttc ccgcctagtg acactgggcc cgcgattcct tggagcgggt
     1741 tgatgacgtc agcgttcgaa ttctaccggg taggggaggc gcttttccca aggcagtctg
     1801 gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc
     1861 gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg
     1921 cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt
     1981 gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg
     2041 ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg
     2101 ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca
     2161 ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc
     2221 gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcagcccaa
     2281 gctagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     2341 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     2401 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     2461 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     2521 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     2581 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     2641 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     2701 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     2761 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     2821 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     2881 ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc
     2941 atgcatcgat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
     3001 ccacctgtag gtttggcaag ctagagaacc atcagatgtt tccagggtgc cccaaggacc
     3061 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
     3121 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
     3181 ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
     3241 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
     3301 gcgggggtct ttcatgggta acagtttctt gaagttggag aacaacattc tgagggtagg
     3361 agtcgaatat taagtaatcc tgactcaatt agccactgtt ttgaatccac atactccaat
     3421 actcctgaaa tagttcatta tggacagcgc agaaagagct ggggagaatt gtgaaattgt
     3481 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3541 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3601 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3661 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3721 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3781 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3841 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3901 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3961 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4021 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4081 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4141 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4201 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4261 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4321 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4381 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4441 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4501 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4561 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4621 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4681 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4741 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4801 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4861 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4921 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4981 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5041 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5101 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5161 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5221 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5281 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5341 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5401 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5461 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5521 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5581 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     5641 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     5701 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     5761 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     5821 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     5881 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
     5941 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     6001 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     6061 acggcgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6121 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6181 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6241 cggccacgat gcgtccggcg tagaggcgat tagtccaatt tgttaaagac aggatatcag
     6301 tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata gagtacgagc
     6361 catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca
     6421 cc
//
