LOCUS       sequence-246630-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes L.monocytogenes CRISPR Type I-B Cas5 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 6172)
  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 6172)
  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-LmoCas5
FEATURES             Location/Qualifiers
     source          1..6172
                     /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(1763..1780)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1769..1993
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2039..2467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2126..2145)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2336..2357
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2476..2496)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2481..2810
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2661..2796
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2723..2742
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2877..3671
                     /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(2931..2950)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3541..3560
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3845..3966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3882..3901)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3936..3955
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4015..4031)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4015..4031)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4028..4050)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4039..4055
                     /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(4063..4069,4070..4087,4088..4093))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4108..4129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4246..4263)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4417..5005)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4497..4516)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5176..5967,5968..6036))
                     /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     5799..5818
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6037..6141)
                     /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 tggactataa ggatgatgac gacaagatgg cccccaagaa gaagcgcaaa
      961 gtccgcggga tgaaggctat tagggtccgg ctgtggcagg atctcgtgaa cttcaagaag
     1021 cctaccagct tccaactgaa ggaaacctac cctctgccgc cgtactctac cgtgatcggc
     1081 atggtgcata cactgtgcgg ctttacctcc taccacgaga tgaagatcag cattcaggga
     1141 aagtacttta gcaaggtcaa cgacctcgcg actagatacg aattcaagaa tggctcaaaa
     1201 tacgacgtgg aacgccacca gatcaaagtg gacaaatacg gagtgtcgag aggcgtcagc
     1261 accgtggagc tccttgtgga cctcgaactg ctgctgcaca tcattccgga agatccatcc
     1321 ttggtgccga tcattgaaaa ggcattccgg gaacccatcg agttcccttc cctgggacgc
     1381 agagaggaca tcgccacgat tcagaaggtc gaagtggtgg acgtcgagaa gcgacagccc
     1441 aagaagtcca ccgagatctc cgacggttat aacgcctacg tgccgatcag cttgaccgag
     1501 aacaactcgg tgcatttcaa gtcccacgag tcctcagtgg gacgggataa gctgctgggg
     1561 actcggtacc ttctgactga gaaatacgag cgcgtgaacc acggaaccga aaaggcccct
     1621 aagttcttcc ggaagtggcg gaagaaggac gtcatctact cctcgcgcat cttcgtgtcc
     1681 aagaaggatg tgttcttttt cgacgaggac gattacctgg tgttcatcga agaagaagag
     1741 tgaagtttaa accgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     1801 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1861 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1921 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     1981 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     2041 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2101 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2161 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2221 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2281 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2341 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2401 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2461 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2521 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2581 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2641 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2701 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     2761 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     2821 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     2881 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2941 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3001 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     3061 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3121 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3181 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3241 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3301 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3361 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     3421 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3481 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3541 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3601 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3661 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     3721 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3781 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3841 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3901 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3961 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4021 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4081 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4141 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4201 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4261 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4321 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4381 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4441 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4501 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4561 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4621 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4681 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4741 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4801 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4861 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4921 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4981 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5041 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5101 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5161 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5221 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5281 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5341 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5401 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5461 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5521 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5581 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5641 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5701 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5761 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5821 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5881 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5941 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6001 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6061 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6121 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
