LOCUS       sequence-206096-        5371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mCherry driven by EF1a, empty gRNA scaffold driven by U6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5371)
  AUTHORS   Pflueger C, Tan D, Swain T, Nguyen TV, Pflueger J, Nefzger C, Polo 
            JM, Ford E, Lister R
  TITLE     A modular dCas9-SunTag DNMT3A epigenome editing system overcomes 
            pervasive off-target activity of direct fusion dCas9-DNMT3A 
            constructs.
  JOURNAL   Genome Res. 2018 Jun 15. pii: gr.233049.117. doi: 
            10.1101/gr.233049.117.
  PUBMED    29907613
REFERENCE   2  (bases 1 to 5371)
  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 Res.
            2018 Jun 15. pii: gr.233049.117. doi: 10.1101/gr.233049.117."
COMMENT     Sequence Label: LLP469 pEF1a-mCherry-EMPTY-gRNA
FEATURES             Location/Qualifiers
     source          1..5371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        284..1462
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          515..1453
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1410..1430
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1469..1478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1475..2185
                     /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(1620..1638)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1888..1907
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     intron          2224..2453
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2436..2455
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     polyA_signal    2632..2753
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2669..2688)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2888..2908
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3059..3078
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3217..3233)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3217..3233)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3230..3252)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3241..3257
                     /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(3265..3271,3272..3289,3290..3295))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3310..3331
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3448..3465)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3619..4207)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3699..3718)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4378..5169,5170..5238))
                     /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     5001..5020
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5239..5343)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgtgcaaag atggataaag ttttaaacag agaggaatct
      241 ttgcagctaa tggaccttct aggtcttgaa aggagtggga attggctccg gtgcccgtca
      301 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
      361 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
      421 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
      481 tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg
      541 gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca
      601 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
      661 cgctcaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg
      721 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
      781 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
      841 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
      901 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
      961 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     1021 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     1081 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     1141 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     1201 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg
     1261 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga
     1321 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc
     1381 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt
     1441 tcttccattt caggtgtcgt gacgtacggc caccatggtg agcaagggcg aggaggataa
     1501 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
     1561 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     1621 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     1681 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     1741 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     1801 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     1861 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     1921 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     1981 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     2041 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     2101 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     2161 cggcatggac gagctgtaca agtgatggaa ttaattcgct gtctgcgagg gccagctgtt
     2221 ggggtgagta ctccctctca aaagcgggca tgacttctgc gctaagattg tcagtttcca
     2281 aaaacgagga ggatttgata ttcacctggc ccgcggtgat gcctttgagg gtggccgcgt
     2341 ccatctggtc agaaaagaca atctttttgt tgtcaagctt gaggtgtggc aggcttgaga
     2401 tctggccata cacttgagtg acaatgacat ccactttgcc tttctctcca caggtgtcca
     2461 ctcccaggtc caactgcagg tcgatttaaa tgggaattcc acagcggccg catgtctaga
     2521 taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     2581 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     2641 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     2701 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaatgcgtc
     2761 gagtgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtattg
     2821 tacaaaaaag caggctttaa aggaaccaat tcagtcgact ggatccggta ccaaggtcgg
     2881 gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     2941 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     3001 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     3061 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttaag
     3121 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     3181 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3241 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3301 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3361 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3421 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3481 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3541 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3601 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3661 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3721 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3781 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3841 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3901 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3961 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4021 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4081 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4141 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4201 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4261 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4321 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4381 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4441 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4501 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4561 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4621 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4681 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4741 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4801 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4861 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4921 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4981 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5041 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5101 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5161 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5221 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5281 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5341 gcgcacattt ccccgaaaag tgccacctga c
//
