LOCUS       sequence-296568-        6504 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 6504)
  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 6504)
  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-empty
FEATURES             Location/Qualifiers
     source          1..6504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        424..803
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        804..1007
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     958..978
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1052..1071
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1052..1070
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1112..1121
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1118..1825
                     /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(1263..1281)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1531..1550
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1826..1852
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1926..1943)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1932..2156
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2202..2630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2289..2308)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2499..2520
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2639..2659)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2644..2973
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2824..2959
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2886..2905
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3040..3834
                     /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(3094..3113)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3704..3723
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4008..4129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4045..4064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4099..4118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4178..4194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4178..4194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4191..4213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4202..4218
                     /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(4226..4232,4233..4250,4251..4256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4271..4292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4409..4426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4580..5168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4660..4679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5339..6130,6131..6199))
                     /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     5962..5981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6200..6304)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6379..6398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gagacggata cgtctctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc aattgcatga
      361 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      421 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      481 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      541 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      601 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      661 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      721 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      781 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      841 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      901 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      961 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     1021 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     1081 ctggctagcg tttaaactta agcttgccgg tgccaccatg gtgagcaagg gcgaggagga
     1141 taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa
     1201 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
     1261 cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc
     1321 tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt
     1381 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
     1441 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa
     1501 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg
     1561 ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca
     1621 gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc
     1681 caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc
     1741 ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac
     1801 cggcggcatg gacgagctgt acaagtaccc atacgatgtt ccagattacg cttaagaatt
     1861 ctgcagatat ccagcacagt ggcggccgct cgagtctaga gggcccgttt aaacccgctg
     1921 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1981 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2041 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2101 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2161 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2221 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2281 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2341 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2401 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2461 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2521 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2581 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     2641 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2701 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2761 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2821 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2881 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     2941 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3001 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3061 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3121 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3181 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3241 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3301 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3361 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3421 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3481 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3541 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3601 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3661 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3721 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3781 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3841 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3901 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3961 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4021 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4081 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4141 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4201 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4261 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4321 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4381 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4441 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4501 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4561 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4621 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4681 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4741 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4801 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4861 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4921 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4981 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5041 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5101 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5161 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5221 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5281 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5341 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5401 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5461 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5521 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5581 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5641 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5701 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5761 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5821 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5881 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5941 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6001 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6061 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6121 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6181 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6241 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6301 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6361 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     6421 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     6481 gcaaggcttg accgacttaa ttaa
//
