LOCUS       sequence-246607-        5997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes E.coli CRISPR Type I-E CasE 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 5997)
  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 5997)
  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-EcoCas6
FEATURES             Location/Qualifiers
     source          1..5997
                     /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(1588..1605)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1594..1818
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1864..2292
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1951..1970)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2161..2182
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2301..2321)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2306..2635
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2486..2621
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2548..2567
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2702..3496
                     /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(2756..2775)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3366..3385
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3670..3791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3707..3726)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3761..3780
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3840..3856)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3840..3856)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3853..3875)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3864..3880
                     /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(3888..3894,3895..3912,3913..3918))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3933..3954
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4071..4088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4242..4830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4322..4341)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5001..5792,5793..5861))
                     /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     5624..5643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5862..5966)
                     /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 tgtatctcag taaagtcatc attgccaggg cctggagcag ggatctttac
     1021 caacttcacc agggattatg gcatttattt ccaaacagac cggatgctgc tcgtgatttt
     1081 ctttttcatg ttgagaagcg aaacacacca gaaggctgtc atgttttatt gcagtcagcg
     1141 caaatgcctg tttcaactgc cgttgcgaca gtcattaaaa ctaaacaggt tgaatttcaa
     1201 cttcaggttg gtgttccact ctattttcgg cttcgggcaa atccgatcaa aactattctc
     1261 gacaatcaaa agcgcctgga cagtaaaggg aatattaaac gctgtcgggt tccgttaata
     1321 aaagaagcag aacaaatcgc gtggttgcaa cgtaaattgg gcaatgcggc gcgcgttgaa
     1381 gatgtgcatc ccatatcgga acggccacag tatttttctg gtgatggtaa aagtggaaag
     1441 atccaaacgg tttgctttga aggtgtgctc accatcaacg acgcgccagc gttaatagat
     1501 cttgtacagc aaggtattgg gccagctaaa tcgatgggat gtggcttgct atctttggct
     1561 ccactgtgag tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1621 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1681 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     1741 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     1801 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     1861 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1921 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1981 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2041 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2101 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2161 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2221 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2281 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2341 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2401 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2461 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2521 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     2581 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     2641 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     2701 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     2761 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     2821 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     2881 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     2941 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3001 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3061 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3121 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3181 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3241 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3301 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3361 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3421 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3481 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     3541 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3601 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3661 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3721 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3781 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     3841 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3901 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3961 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4021 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4081 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4141 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4201 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4261 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4321 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4381 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4441 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4501 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4561 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4621 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4681 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4741 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4801 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4861 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4921 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4981 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5041 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5101 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5161 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5221 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5281 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5341 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5401 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5461 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5521 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5581 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5641 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5701 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5761 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5821 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5881 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5941 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
