LOCUS       sequence-246632-        6121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes L.monocytogenes CRISPR Type I-B Cas6 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 6121)
  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 6121)
  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-LmoCas6
FEATURES             Location/Qualifiers
     source          1..6121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(108..128)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        113..442
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      293..428
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     355..374
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             509..1303
                     /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(563..582)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1173..1192
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1477..1598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1514..1533)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1568..1587
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1647..1663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1647..1663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1660..1682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1671..1687
                     /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(1695..1701,1702..1719,1720..1725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1740..1761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1878..1895)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2049..2637)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2129..2148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2808..3599,3600..3668))
                     /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     3431..3450
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3669..3773)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3848..3867)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4039..4418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4419..4622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4573..4593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4667..4686
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4667..4685
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4708..4717
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4717..4740
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4747..4767
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5516..5533)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5522..5746
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(5792..6121,1..99)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5879..5898)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6089..6110
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
       61 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
      121 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
      181 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
      241 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
      301 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
      361 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
      421 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
      481 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
      541 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
      601 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
      661 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
      721 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
      781 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
      841 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
      901 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
      961 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     1021 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     1081 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     1141 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     1201 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     1261 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     1321 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     1381 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     1441 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     1501 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1561 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     1621 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     1681 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     1741 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1801 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1861 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1921 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1981 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2041 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2101 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2161 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2221 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2281 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2341 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2401 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2461 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     2521 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2581 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2641 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2701 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2761 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2821 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2881 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2941 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3001 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3061 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3121 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3181 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3241 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3301 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3361 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3421 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3481 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3541 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3601 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3661 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3721 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3781 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     3841 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     3901 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     3961 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     4021 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     4081 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     4141 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     4201 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     4261 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     4321 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     4381 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     4441 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     4501 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     4561 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     4621 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     4681 atagggagac ccaagctggc tagcgccgcc accatggact acaaggacga cgacgataag
     4741 atggcgccaa agaaaaagcg caaggtccgc ggaatgcggc tgaagattaa ctgcgacttc
     4801 gacagcaaga tcatccccaa ggacttccag tccaaggtcg tgtccctgtt caaagccgga
     4861 attatgaaaa gctccccgga gagatacgag aacctgtttg gtggaaacaa gcacaagcag
     4921 tacactttct cggtgtatct cccaaaacct cagaacaagg gcgccgaaat tcagctgaac
     4981 gaggcgaact gtatcatcaa cttctccacc ggcgatgctg aaaccggtat tatcttctac
     5041 aacgccctga ttagcctgag aggctccaag gtcctgtttg gggccggaaa tcacatcacc
     5101 gtgaagaaca tccagatcgt gcctgagaag aagatcatcg ggaagcaaac cattctgcgg
     5161 accctttcgc cggtggtgtc gagggatcac aaccgggaaa ccttcaagaa ttggttctac
     5221 tcattcgagg acgaagaatt cgagcctact ttgaagcgga acatgctgcc gtacctcatc
     5281 gacgcctttg gagaacaggc tcgctacgat ctggaaaagc tgaagatcac gcccatttcc
     5341 atgaagaaag tcgtggtgta ctgccatgag atccacatcg agagctccgt gggcatcttc
     5401 gagctggaag ccgaggcata cctccaaaag tacctcgtgg aaaacggaat cggcactatg
     5461 actggctcag ggttcggaat gattgaacag ttctgaagtt taaaccgctg atcagcctcg
     5521 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     5581 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     5641 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     5701 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     5761 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     5821 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5881 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5941 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6001 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     6061 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     6121 a
//
