LOCUS       sequence-246603-        5880 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes human codon optimized E.coli CRISPR Type I-E CasB 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 5880)
  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 5880)
  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-EcoCse2
FEATURES             Location/Qualifiers
     source          1..5880
                     /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(1471..1488)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1477..1701
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1747..2175
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1834..1853)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2044..2065
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2184..2204)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2189..2518
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2369..2504
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2431..2450
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2585..3379
                     /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(2639..2658)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3249..3268
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3553..3674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3590..3609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3644..3663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3723..3739)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3723..3739)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3736..3758)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3747..3763
                     /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(3771..3777,3778..3795,3796..3801))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3816..3837
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3954..3971)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4125..4713)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4205..4224)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4884..5675,5676..5744))
                     /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     5507..5526
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5745..5849)
                     /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 gtacgcggga tggccgatga aatagacgca atggctcttt accgagcatg gcaacagctc
     1021 gacaatggat cttgcgccca aatacggcgg gtaagtgaac ccgatgaact gcgagatatc
     1081 cccgcattct accgattggt tcagccgttt ggctgggaga acccacggca ccagcaggcg
     1141 cttcttagga tggttttttg tcttagtgcc gggaaaaacg taatccgcca tcaggataag
     1201 aagtccgaac aaacaacagg gatttctctg ggaagagcgc ttgctaacag cggcaggatc
     1261 aatgaacgcc gcatatttca gttgatccga gcagatcgga ctgctgatat ggtccagctc
     1321 aggaggctcc ttacgcacgc agagccagtg ttggattggc cactcatggc aagaatgctt
     1381 acgtggtggg ggaagaggga aaggcagcaa ctgcttgaag attttgtatt gacgacgaac
     1441 aaaaacgcgt aagtttaaac cgctgatcag cctcgactgt gccttctagt tgccagccat
     1501 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1561 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1621 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1681 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     1741 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1801 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1861 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1921 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1981 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2041 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2101 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2161 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2221 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2281 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2341 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2401 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     2461 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     2521 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     2581 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     2641 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     2701 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     2761 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     2821 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     2881 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     2941 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3001 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3061 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     3121 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3181 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3241 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3301 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3361 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     3421 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3481 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3541 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3601 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3661 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     3721 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3781 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3841 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3901 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3961 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4021 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4081 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4141 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4201 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4261 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4321 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4381 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4441 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4501 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4561 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4621 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4681 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4741 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4801 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4861 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4921 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4981 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5041 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5101 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5161 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5221 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5281 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5341 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5401 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5461 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5521 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5581 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5641 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5701 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5761 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5821 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
