LOCUS       sequence-246628-        7087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes L.monocytogenes CRISPR Type I-B Cas8b2 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 7087)
  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 7087)
  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-LmoCas8b2
FEATURES             Location/Qualifiers
     source          1..7087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..34)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        19..348
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      199..334
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     261..280
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             415..1209
                     /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(469..488)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1079..1098
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1383..1504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1420..1439)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1474..1493
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1553..1569)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1553..1569)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1566..1588)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1577..1593
                     /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(1601..1607,1608..1625,1626..1631))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1646..1667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1784..1801)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1955..2543)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2035..2054)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2714..3505,3506..3574))
                     /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     3337..3356
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3575..3679)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3754..3773)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3945..4324
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4325..4528
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4479..4499
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4573..4592
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4573..4591
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4614..4623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4623..4646
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4653..4673
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6388..6405)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6394..6618
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6664..7087,1..5)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6751..6770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6961..6982
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
       61 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
      121 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
      181 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
      241 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
      301 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
      361 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
      421 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
      481 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
      541 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
      601 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
      661 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
      721 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
      781 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
      841 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
      901 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
      961 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     1021 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     1081 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     1141 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     1201 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     1261 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     1321 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     1381 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1441 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1501 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     1561 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1621 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1681 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1741 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1801 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1861 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1921 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1981 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2041 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2101 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2161 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2221 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2281 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2341 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2401 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2461 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2521 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2581 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     2641 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     2701 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     2761 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     2821 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     2881 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     2941 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3001 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3061 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3121 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3181 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3241 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3301 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3361 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3421 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3481 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3541 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     3601 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3661 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     3721 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3781 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     3841 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     3901 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     3961 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     4021 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     4081 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     4141 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     4201 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     4261 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     4321 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     4381 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     4441 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     4501 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
     4561 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gccgccacca
     4621 tggattacaa ggacgatgac gacaaaatgg caccgaagaa gaagcggaag gtccgcggaa
     4681 tgcagaccga aatcgaagtc agggcagacg actggctcat caacaccgga ctgaccggct
     4741 tcctgaacat tgtgggaaaa gagaacgtcc ggattgacgg gcagtctatc tacttcaccg
     4801 ctgacctcct ggaagatttc gaaactaagt actttaactt cttcatccaa gagtacaaag
     4861 aaactctctc atggtacaag atcgtcagct acaaagagac tatggagcag ttcagaattg
     4921 atgaattcgc gtcctttgac gaagtggccc tggggaacct gaacaagtac atgaaggacg
     4981 tcgtgaagtt ctatctgaaa aaggccaact atatcaaagt gttccccttg attgacccta
     5041 gcgccaacgt gaccaagtgg ctcgaaaagc tgaacaccat taatctgacc aagaagcaga
     5101 aatgggatga ggtcaaggct gacattattg aggaggtcaa acagacctac acccaactcg
     5161 acctgattat tgacttctgt gcctccgaaa agggactgca gtacctggga gccaagaacc
     5221 tgatctattc cgtcattaac aagggctgga gcggagtctc cttcctgttc aagcaaacca
     5281 agttcattga cccgtatctg gactacaaga cgaccttcgt ggatcctgtg ctggagtacc
     5341 tggataccga tctgtccaag gccaagtaca actgcttcaa ctgcaaccag cccatcaaga
     5401 acctcaagct cgacctctcg ttcatgaatg acgtgggctt tgacaccgcc cgcaagactg
     5461 cgcacgtctg ggatttcaac aacgatgtgg ccacttgtcc aatctgccgg ctgatctact
     5521 catgcgtgcc tgccgggttc acctacgtgt atggagaggg aatgttcgtg aacgactcct
     5581 acggcgtgga agaactgcac agagtgaacg aacggatgcg gaacagcatc ctgaggttta
     5641 acaaggacgg tatcaattcc accaacactt accgcgcgct tgtggagtcc attaccatgg
     5701 aacacgaaaa caacagacgc tacgaattgg ctgacatcca gctcgtgcga tacgagaacg
     5761 agcattaccg gttcaatctg ttgtcaaaga agatgcttca catcgtgaac gattccaagg
     5821 gtatcctgaa gtcgctgata cgctgcggtt acaaggaggg gaacctcaac gtgaacctct
     5881 acaaggaagt gatccagcat ctgatgaaca acgaaaacct cttcaccctt atccacaagt
     5941 tgatctacta caagcagacc agcgtgaacg gcctgtacta caacatgggc cacgtgtccg
     6001 gaatcctgga tatcaacacc aagttcctga aagaaatgga ggtcatgacg aacatctcac
     6061 agaaccagct gtggttcgtg caaaattgtg gcaaagaatt caaggagggt tacgtgaaga
     6121 agaagtccga gaacaagatc tccggaatca cttacaagct gctgaattcg ctgaaggtca
     6181 acgacaagga cggattcatg gacaccctgc tgaactccta ctcgtacctg tccatgccga
     6241 tccccgacgt gttcatcaag atgttctcca acaacgaagc cttcaagagc gtgggctacg
     6301 cttttatgct tggcgtgggt ggagaaaaga ctaagaagga agatgggggc aacaccgatg
     6361 agaagtgaag tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     6421 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     6481 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     6541 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     6601 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     6661 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     6721 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     6781 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     6841 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     6901 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     6961 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     7021 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     7081 ttaacgc
//
