LOCUS       sequence-412379-        5187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression vector with mCherry transfection control, does not 
            contain the direct repeat of the truncated guideRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5187)
  AUTHORS   Kulcsar PI, Talas A, Toth E, Nyeste A, Ligeti Z, Welker Z, Welker E
  TITLE     Blackjack mutations improve the on-target activities of increased 
            fidelity variants of SpCas9 with 5'G-extended sgRNAs.
  JOURNAL   Nat Commun. 2020 Mar 6;11(1):1223. doi: 10.1038/s41467-020-15021-5.
  PUBMED    32144253
REFERENCE   2  (bases 1 to 5187)
  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: "2020-03-6"; volume: "11(1):1223. doi"; pages: " 
            10.1038/s41467-020-15021-5"
COMMENT     Sequence Label: pmCherry_sgRNA-ver2
FEATURES             Location/Qualifiers
     source          1..5187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          273..500
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      512..521
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             518..1228
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(663..681)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    1357..1478
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1394..1413)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1448..1467
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1608..1627
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1741..1845
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1847..2204
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1847..2038
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1870..1890)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2055..2190
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2239..3033
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2239..3033
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2293..2312)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2903..2922
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3221..3240)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3265..3312
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3641..4229
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4130..4149
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4291..4539
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4291..4311
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4462..4481
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4568..4643
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        4662..4947
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gcatgtgcgt ggtgttgagc atccctccgc caaccgcaca ccttcaccta tagcgttgtc
       61 tgtaattatt ttgtgcagcg atgggggcgg gggggggggg gggggggggg gggggggggg
      121 cgcggggggg gggggggggg ggcggggggc gggggggggc gaggcggaga ggtgcggcgg
      181 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      241 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
      301 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
      361 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
      421 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
      481 ctgaaatcac tttttttcag gttggaccgg tgccaccatg gtgagcaagg gcgaggagga
      541 taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa
      601 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
      661 cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc
      721 tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt
      781 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
      841 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa
      901 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaaaaaga ccatgggctg
      961 ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca
     1021 gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc
     1081 caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc
     1141 ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac
     1201 cggcggcatg gacgagctgt acaagtagga tccaccggat ctagataact gatcataatc
     1261 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     1321 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     1381 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1441 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcggtgaacc atcaccctaa
     1501 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     1561 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     1621 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     1681 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     1741 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1801 caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga
     1861 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     1921 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     1981 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2041 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     2101 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     2161 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca
     2221 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     2281 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     2341 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     2401 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     2461 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     2521 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     2581 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2641 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     2701 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     2761 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     2821 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     2881 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     2941 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3001 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     3061 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     3121 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3181 tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
     3241 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg
     3301 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     3361 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     3421 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct
     3481 caggttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3541 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3601 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3661 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3721 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3781 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3841 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3901 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3961 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4021 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4081 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4141 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4201 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4261 tggccttttg ctggcctttt gctcacatgt gagggcctat ttcccatgat tccttcatat
     4321 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     4381 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     4441 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     4501 ttcttggctt tatatatctt gtggaaagga cgaaacaccg ggtcttcgga tccagatctg
     4561 aagacctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     4621 aaaaagtggc accgagtcgg tgcttttttt ctagaggtac ccgttacata acttacggta
     4681 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca
     4741 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     4801 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     4861 cccgcctggc attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     4921 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     4981 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
     5041 gcagcgatgg gggcgggggg gggggggggg cggggcccgg gggggggggg gggggggggg
     5101 gggggggggg gggggggggc gggggggggg ggggcggggg cccagggggg gggggggggg
     5161 ggggttcttt tatggggagg gggggcg
//
