LOCUS       sequence-151482-        5033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression vector with mCherry transfection control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5033)
  AUTHORS   Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor 
            E, Welker E
  TITLE     Crossing enhanced and high fidelity SpCas9 nucleases to optimize 
            specificity and cleavage.
  JOURNAL   Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8.
  PUBMED    28985763
REFERENCE   2  (bases 1 to 5033)
  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: "Genome 
            Biol."; date: "2017-10-6"; volume: "18(1):190. doi"; pages: " 
            10.1186/s13059-017-1318-8"
COMMENT     Sequence Label: pmCherry_gRNA
FEATURES             Location/Qualifiers
     source          1..5033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        284..359
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        456..741
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        743..1020
                     /label=chicken beta-actin promoter
     intron          1021..1249
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1261..1270
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1267..1977
                     /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(1412..1430)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    2106..2227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2143..2162)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2197..2216
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2357..2376
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2490..2594
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2596..2953
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2596..2787
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2619..2639)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2804..2939
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2988..3782
                     /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             2988..3782
                     /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(3042..3061)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3652..3671
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3970..3989)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4014..4061
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4390..4978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4879..4898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccgggtc ttcggatcca gatctgaaga cctgttttag agctagaaat
      301 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
      361 tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg
      421 cgccaattct gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc
      481 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg
      541 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      601 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      661 tggcattgtg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      721 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
      781 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
      841 atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
      901 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      961 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     1021 ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     1081 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     1141 tccgggctgt aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt
     1201 attaatgttt aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt
     1261 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     1321 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1381 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1441 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1501 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1561 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1621 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1681 cccgtaatgc agaaaaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     1741 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     1801 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1861 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     1921 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaggat
     1981 ccaccggatc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     2041 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     2101 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2161 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2221 tatcttaacg cggtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     2281 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2341 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2401 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2461 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2521 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2581 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     2641 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2701 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2761 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     2821 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2881 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2941 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3001 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3061 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3121 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     3181 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3241 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3301 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3361 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3421 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3481 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     3541 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3601 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3661 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3721 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     3781 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     3841 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     3901 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     3961 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     4021 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     4081 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     4141 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     4201 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     4261 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4321 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4381 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4441 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4501 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4561 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4621 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4681 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4741 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4801 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4861 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4921 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4981 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctc
//
