LOCUS       sequence-297949-        6506 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 6506)
  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 6506)
  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-ALDH1A3
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        85..333
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     85..105
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     256..275
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        354..429
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        510..889
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        890..1093
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1044..1064
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1138..1157
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1138..1156
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1198..1207
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1204..1911
                     /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(1349..1367)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1617..1636
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1912..1938
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2012..2029)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2018..2242
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2288..2716
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2375..2394)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2585..2606
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2725..2745)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2730..3059
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2910..3045
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2972..2991
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3126..3920
                     /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(3180..3199)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3790..3809
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4094..4215
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4131..4150)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4185..4204
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4264..4280)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4264..4280)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4277..4299)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4288..4304
                     /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(4312..4318,4319..4336,4337..4342))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4357..4378
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4495..4512)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4666..5254)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4746..4765)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5425..6216,6217..6285))
                     /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     6048..6067
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6286..6390)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6465..6484)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
       61 gcaaggcttg accgacttaa ttaagagggc ctatttccca tgattccttc atatttgcat
      121 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      181 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      241 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      301 gctttatata tcttgtggaa aggacgaaac accggagagg gaccgcgcca cctgttttag
      361 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      421 agtcggtgct tttttcaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
      481 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      541 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      601 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      661 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      721 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      781 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      841 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      901 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      961 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1021 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1081 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     1141 tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tgccggtgcc
     1201 accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
     1261 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     1321 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     1381 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
     1441 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     1501 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     1561 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     1621 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     1681 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     1741 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     1801 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     1861 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gtacccatac
     1921 gatgttccag attacgctta agaattctgc agatatccag cacagtggcg gccgctcgag
     1981 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2041 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2101 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2161 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2221 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2281 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2341 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2401 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2461 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2521 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2581 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2641 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2701 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     2761 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     2821 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2881 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     2941 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3001 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3061 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     3121 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3181 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3241 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3301 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3361 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3421 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3481 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     3541 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3601 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     3661 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     3721 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     3781 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     3841 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     3901 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     3961 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4021 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4081 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4141 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4201 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4261 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4321 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4381 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4441 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4501 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4561 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4621 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4681 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4741 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4801 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4861 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4921 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4981 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5041 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5101 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5161 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5221 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5281 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5341 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5401 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5461 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5521 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5581 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5641 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5701 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5761 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5821 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5881 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5941 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6001 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6061 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6121 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6181 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6241 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6301 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6361 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6421 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     6481 gccgcatagt taagccagta tctgct
//
