LOCUS       sequence-246604-        6909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes human codon optimized E.coli CRISPR Type I-E CasA with 
            N-terminal fusion of Flag tag epitope and nuclear localization 
            signal driven by CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6909)
  AUTHORS   Pickar-Oliver A, Black JB, Lewis MM, Mutchnick KJ, Klann TS, 
            Gilcrest KA, Sitton MJ, Nelson CE, Barrera A, Bartelt LC, Reddy TE, 
            Beisel CL, Barrangou R, Gersbach CA
  TITLE     Targeted transcriptional modulation with type I CRISPR-Cas systems 
            in human cells.
  JOURNAL   Nat Biotechnol. 2019 Sep 23. pii: 10.1038/s41587-019-0235-7. doi: 
            10.1038/s41587-019-0235-7.
  PUBMED    31548729
REFERENCE   2  (bases 1 to 6909)
  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: "Nat 
            Biotechnol. 2019 Sep 23. pii: 10.1038/s41587-019-0235-7. doi: 
            10.1038/s41587-019-0235-7."
COMMENT     Sequence Label: pcDNA-Flag-EcoCas8e
FEATURES             Location/Qualifiers
     source          1..6909
                     /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"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      904..913
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             913..936
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             943..963
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2500..2517)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2506..2730
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2776..3204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2863..2882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3073..3094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3213..3233)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3218..3547
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3398..3533
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3460..3479
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3614..4408
                     /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(3668..3687)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4278..4297
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4582..4703
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4619..4638)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4673..4692
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4752..4768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4752..4768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4765..4787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4776..4792
                     /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(4800..4806,4807..4824,4825..4830))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4845..4866
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4983..5000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5154..5742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5234..5253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5913..6704,6705..6773))
                     /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     6536..6555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6774..6878)
                     /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 caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccgccacca tggattacaa agacgatgac gataagatgg cgcctaagaa gaagcgcaag
      961 gtgcggggca tggccaatct cctgattgac aattggatcc ctgtgagacc gaggaacgga
     1021 gggaaggttc agatcatcaa cctgcaaagt ctctactgta gcagagatca atggcgactc
     1081 tcattgccca gggatgatat ggaattggcg gcacttgcac tcttggtatg tattgggcag
     1141 atcatcgccc ctgccaagga tgatgtcgag tttaggcaca gaataatgaa ccctcttact
     1201 gaagatgaat tccagcaatt gatcgcgcct tggatagata tgttctattt gaatcacgcc
     1261 gaacatccct tcatgcagac aaaaggggtt aaagcaaatg atgtcacacc aatggagaag
     1321 cttttggcag gtgtgtcagg agcaaccaac tgcgctttcg ttaaccaacc aggccaaggt
     1381 gaggcgttgt gcggggggtg cacggccata gcgttgttta atcaggcgaa ccaggctcct
     1441 ggctttggag gaggattcaa gtccggtttg cgcgggggta cacccgtaac cacattcgtt
     1501 cgcggaattg acctccgatc aacagtcctg ttgaacgtat tgacgttgcc tagacttcag
     1561 aagcaatttc caaacgagtc tcacactgag aatcaaccga cttggattaa gcccatcaag
     1621 tcaaatgaaa gtatacccgc gtcttctatt ggatttgtta gaggactttt ttggcagccc
     1681 gctcacatag aactctgtga tcccataggc attggaaagt gttcttgctg tggccaagag
     1741 tctaacctta gatatacggg ctttctcaag gagaagttca cgttcaccgt gaatggactt
     1801 tggccacatc cccatagccc gtgcttggtc acggtcaaga agggggaggt tgaagagaaa
     1861 tttcttgcgt ttaccacatc cgccccatcc tggacgcaaa tatctcgcgt cgttgtggat
     1921 aaaatcatcc agaacgaaaa tggtaaccgc gttgccgcgg tagtgaatca attcagaaac
     1981 atagcaccac agagcccgtt ggagttgata atgggcgggt accgcaataa tcaagcttcc
     2041 attctcgagc gacgccatga cgtacttatg tttaaccagg ggtggcagca atatggtaac
     2101 gtgataaacg agatagtcac agtcggcctc ggctacaaaa ctgcgttgcg caaagctctg
     2161 tatacgttcg cggagggttt taagaataaa gactttaaag gcgctggggt gagtgtgcac
     2221 gagacggcag aacgccattt ctatcgccag tctgaactct tgattcccga cgttctggcg
     2281 aatgtcaatt tttcccaggc cgatgaggtc attgcggatc tgcgagataa gcttcaccag
     2341 ttgtgtgaga tgctgttcaa ccagtctgtg gctccttatg ctcatcatcc caaactgatt
     2401 tcaactctcg ctttggcaag ggctacgttg tataaacacc ttagagaact gaagccacag
     2461 ggtggtccca gcaatggctg agtttaaacc gctgatcagc ctcgactgtg ccttctagtt
     2521 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2581 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2641 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2701 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2761 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2821 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2881 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2941 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3001 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3061 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3121 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3181 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3241 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3301 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3361 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3421 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3481 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3541 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     3601 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3661 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3721 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3781 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3841 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3901 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     3961 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4021 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4081 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4141 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4201 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4261 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4321 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4381 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4441 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4501 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4561 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4621 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4681 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4741 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4801 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4861 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4921 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4981 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5041 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5101 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5161 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5221 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5281 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5341 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5401 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5461 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5521 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5581 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5641 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5701 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5761 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5821 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5881 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5941 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6001 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6061 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6121 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6181 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6241 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6301 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6361 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6421 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6481 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6541 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6601 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6661 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6721 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6781 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6841 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6901 cctgacgtc
//
