LOCUS       sequence-272810-        6503 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a gRNA and mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6503)
  AUTHORS   Schmid-Burgk JL, Gao L, Li D, Gardner Z, Strecker J, Lash B, Zhang F
  TITLE     Highly Parallel Profiling of Cas9 Variant Specificity.
  JOURNAL   Mol Cell. 2020 Mar 17. pii: S1097-2765(20)30143-X. doi: 
            10.1016/j.molcel.2020.02.023.
  PUBMED    32187529
REFERENCE   2  (bases 1 to 6503)
  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: "Mol Cell. 
            2020 Mar 17. pii: S1097-2765(20)30143-X. doi: 
            10.1016/j.molcel.2020.02.023."
COMMENT     Sequence Label: pmCherry-U6-CACNG3
FEATURES             Location/Qualifiers
     source          1..6503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        170..418
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     170..190
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     341..360
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        439..514
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        595..974
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        975..1178
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1129..1149
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1223..1242
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1223..1241
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1283..1292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1289..1996
                     /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(1434..1452)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1702..1721
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1997..2023
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2097..2114)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2103..2327
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2373..2801
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2460..2479)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2670..2691
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2810..2830)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2815..3144
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2995..3130
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3057..3076
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3211..4005
                     /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(3265..3284)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3875..3894
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4179..4300
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4216..4235)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4270..4289
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4349..4365)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4349..4365)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4362..4384)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4373..4389
                     /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(4397..4403,4404..4421,4422..4427))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4442..4463
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4580..4597)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4751..5336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4831..4850)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5507..6298,6299..6367))
                     /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     6130..6149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6368..6472)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga cttaattaag agggcctatt
      181 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
      241 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      301 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      361 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
      421 acttacgcag gagatattgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
      481 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt caattgcatg aagaatctgc
      541 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      601 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      661 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      721 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      781 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      841 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      901 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      961 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     1021 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     1081 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     1141 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
     1201 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
     1261 gtttaaactt aagcttgccg gtgccaccat ggtgagcaag ggcgaggagg ataacatggc
     1321 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     1381 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     1441 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     1501 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     1561 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     1621 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     1681 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     1741 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     1801 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     1861 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     1921 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     1981 ggacgagctg tacaagtacc catacgatgt tccagattac gcttaagaat tctgcagata
     2041 tccagcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     2101 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2161 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2221 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2281 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2341 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2401 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2461 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2521 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2581 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2641 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2701 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2761 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2821 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2881 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2941 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3001 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3061 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3121 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     3181 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     3241 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3301 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3361 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     3421 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3481 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     3541 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3601 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3661 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3721 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3781 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3841 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3901 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3961 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4021 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4081 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4141 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4201 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4261 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4321 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4381 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4441 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4501 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4561 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4621 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4681 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4741 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4801 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4861 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4921 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4981 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5041 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5101 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5161 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5221 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5281 gtagcggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5341 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5401 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5461 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5521 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5581 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5641 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5701 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5761 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5821 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5881 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5941 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6001 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6061 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6121 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6181 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6241 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6301 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6361 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6421 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6481 cccgaaaagt gccacctgac gtc
//
