LOCUS       sequence-246606-        6072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes human codon optimized E.coli CRISPR Type I-E CasD 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 6072)
  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 6072)
  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-EcoCas5
FEATURES             Location/Qualifiers
     source          1..6072
                     /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(1663..1680)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1669..1893
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1939..2367
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2026..2045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2236..2257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2376..2396)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2381..2710
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2561..2696
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2623..2642
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2777..3571
                     /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(2831..2850)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3441..3460
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3745..3866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3782..3801)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3836..3855
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3915..3931)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3915..3931)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3928..3950)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3939..3955
                     /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(3963..3969,3970..3987,3988..3993))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4008..4029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4146..4163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4317..4905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4397..4416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5076..5867,5868..5936))
                     /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     5699..5718
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5937..6041)
                     /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 aaagaggaaa
      961 gtacggggca tgcgatcata cttgatcctg cggcttgcag gtcctatgca agcctggggg
     1021 caacctacct ttgaaggtac tcggccgact ggcaggttcc ctacgcggtc tggtttgctc
     1081 ggactcctcg gcgcctgttt ggggatacaa agggatgaca cttcttcctt gcaggcactt
     1141 tccgaatcag tccagttcgc agtgagatgt gatgaactca tactggacga cagacgggtg
     1201 tccgtaactg gactgaggga ctatcatact gtactcggcg caagagaaga ttatcgaggt
     1261 cttaagtcac atgagactat tcagacatgg agggaatatt tgtgtgacgc ctccttcacg
     1321 gtggccctct ggcttacacc acatgcaact atggtgatct cagagcttga gaaagccgtt
     1381 cttaaacctc ggtacacacc atatctgggg aggcggtctt gcccacttac ccacccgctt
     1441 ttcttgggga cttgtcaggc cagcgatcca cagaaggcct tgctgaacta tgaacccgtt
     1501 ggtggcgata tatacagtga agagagcgtc acgggccatc acttgaagtt cactgctagg
     1561 gatgagccga tgattacgct cccgagacag ttcgctagta gggaatggta cgttattaag
     1621 gggggaatgg acgtttccca atgagtttaa accgctgatc agcctcgact gtgccttcta
     1681 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1741 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1801 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     1861 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     1921 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1981 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2041 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2101 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2161 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2221 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2281 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2341 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2401 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2461 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2521 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2581 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2641 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2701 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     2761 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2821 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2881 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2941 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3001 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3061 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3121 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3181 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3241 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3301 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3361 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3421 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3481 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3541 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     3601 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3661 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3721 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3781 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3841 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3901 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3961 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4021 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4081 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4141 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4201 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4261 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4321 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4381 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4441 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4501 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4561 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4621 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4681 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4741 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4801 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4861 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4921 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4981 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5041 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5101 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5161 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5221 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5281 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5341 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5401 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5461 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5521 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5581 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5641 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5701 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5761 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5821 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5881 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5941 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6001 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6061 ccacctgacg tc
//
