LOCUS       sequence-246605-        6491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes human codon optimized E.coli CRISPR Type I-E CasC 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 6491)
  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 6491)
  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-EcoCas7
FEATURES             Location/Qualifiers
     source          1..6491
                     /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(2082..2099)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2088..2312
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2358..2786
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2445..2464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2655..2676
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2795..2815)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2800..3129
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2980..3115
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3042..3061
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3196..3990
                     /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(3250..3269)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3860..3879
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4164..4285
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4201..4220)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4255..4274
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4334..4350)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4334..4350)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4347..4369)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4358..4374
                     /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(4382..4388,4389..4406,4407..4412))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4427..4448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4565..4582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4736..5324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4816..4835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5495..6286,6287..6355))
                     /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     6118..6137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6356..6460)
                     /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 gtgagaggaa tgtcaaactt cattaatatc cacgtgctta tctcacactc ccctagttgc
     1021 cttaacagag acgatatgaa catgcaaaaa gacgcaattt ttggcggcaa aaggagagtc
     1081 agaattagta gccagagcct gaagcgcgct atgaggaaaa gcggctacta tgctcaaaac
     1141 attggtgaaa gttcattgcg gaccatccat ctcgcgcagt tgagggacgt cctgcgacag
     1201 aagcttgggg aaagatttga tcagaagatc atcgacaaaa cgcttgccct tctgtccggt
     1261 aaatcagtgg acgaagcgga gaagataagt gcggatgctg ttacgccatg ggtggtaggt
     1321 gaaatcgcgt ggttttgcga gcaggtagcc aaggccgaag cggataattt ggatgataag
     1381 aaactgctca aagtcctcaa agaggacatc gcggcgatcc gggtgaacct tcagcagggt
     1441 gttgatattg cgctctctgg tcggatggcc acgtctggaa tgatgactga actgggtaag
     1501 gtggacggag ctatgtctat agctcatgct ataactaccc atcaggtgga ttctgacata
     1561 gactggttca cggctgtcga cgatctccag gaacaaggat ccgcacacct cggcacgcaa
     1621 gaattttctt ctggagtgtt ctataggtat gccaacatca accttgcaca gctccaggaa
     1681 aacctcggtg gggcaagccg ggaacaggct cttgaaatag ctacccatgt ggttcacatg
     1741 ctggcgaccg aagtgccagg ggccaagcag agaacgtacg ccgcattcaa tccggcggac
     1801 atggtcatgg tgaatttctc tgatatgccc ttgtctatgg caaatgcttt cgagaaggcg
     1861 gtcaaggcaa aggacggttt tttgcaaccc tccatccaag cctttaatca gtactgggat
     1921 agagtagcta acgggtatgg tctcaatggc gcggctgctc agttttcttt gtccgatgtg
     1981 gatccgataa cggcgcaggt taaacagatg cccaccttgg aacaactcaa atcctgggtt
     2041 agaaacaatg gggaggcgtg agagtttaaa ccgctgatca gcctcgactg tgccttctag
     2101 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2161 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2221 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2281 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2341 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2401 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2461 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2521 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2581 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2641 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2701 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2761 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     2821 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2881 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2941 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3001 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3061 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3121 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3181 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3241 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3301 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3361 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3421 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3481 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3541 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3601 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3661 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3721 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3781 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3841 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3901 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3961 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4021 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4081 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4141 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4201 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4261 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4321 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4381 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4441 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4501 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4561 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4621 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4681 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4741 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4801 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4861 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4921 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4981 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5041 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5101 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5161 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5221 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5281 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5341 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5401 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5461 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5521 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5581 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5641 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5701 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5761 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5821 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5881 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5941 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6001 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6061 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6121 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6181 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6241 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6301 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6361 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6421 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6481 cacctgacgt c
//
