LOCUS       sequence-296496-        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-CLIC3
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(180..199)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        306..554
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     306..326
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     477..496
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        575..650
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        731..1110
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1111..1314
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1265..1285
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1359..1378
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1359..1377
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1419..1428
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1425..2132
                     /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(1570..1588)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1838..1857
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2133..2159
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2233..2250)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2239..2463
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2509..2937
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2596..2615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2806..2827
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2946..2966)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2951..3280
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3131..3266
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3193..3212
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3347..4141
                     /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(3401..3420)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4011..4030
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4315..4436
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4352..4371)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4406..4425
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4485..4501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4485..4501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4498..4520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4509..4525
                     /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(4533..4539,4540..4557,4558..4563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4578..4599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4716..4733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4887..5475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4967..4986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5646..6437,6438..6506))
                     /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     6269..6288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
      181 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
      241 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgactta
      301 attaagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
      361 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      421 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      481 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
      541 aaggacgaaa caccgacaga cacgctgcag atcggtttta gagctagaaa tagcaagtta
      601 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttcaat
      661 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      721 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      781 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      841 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      901 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      961 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     1021 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     1081 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     1141 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     1201 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     1261 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
     1321 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
     1381 acccaagctg gctagcgttt aaacttaagc ttgccggtgc caccatggtg agcaagggcg
     1441 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
     1501 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1561 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1621 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1681 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1741 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     1801 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     1861 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     1921 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1981 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     2041 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     2101 actccaccgg cggcatggac gagctgtaca agtacccata cgatgttcca gattacgctt
     2161 aagaattctg cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa
     2221 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2281 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2341 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2401 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2461 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2521 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2581 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2641 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2701 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2761 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2821 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2881 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     2941 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3001 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3061 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3121 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3181 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3241 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3301 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3361 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3421 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3481 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3541 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3601 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3661 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3721 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3781 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3841 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3901 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3961 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4021 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4081 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4141 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4201 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4261 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4321 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4381 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4441 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4501 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4561 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4621 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4681 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4741 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4801 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4861 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4921 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4981 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5041 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5101 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5161 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5221 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5281 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5341 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5401 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5461 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5521 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5581 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5641 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5701 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5761 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5821 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5881 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5941 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6001 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6061 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6121 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6181 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6241 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6301 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6361 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6421 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6481 gcgacacgga aatgttgaat actcat
//
