LOCUS       sequence-246629-        6268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes L.monocytogenes CRISPR Type I-B Cas7 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 6268)
  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 6268)
  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-LmoCas7
FEATURES             Location/Qualifiers
     source          1..6268
                     /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(1859..1876)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1865..2089
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2135..2563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2222..2241)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2432..2453
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2572..2592)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2577..2906
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2757..2892
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2819..2838
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2973..3767
                     /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(3027..3046)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3637..3656
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3941..4062
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3978..3997)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4032..4051
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4111..4127)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4111..4127)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4124..4146)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4135..4151
                     /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(4159..4165,4166..4183,4184..4189))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4204..4225
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4342..4359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4513..5101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4593..4612)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5272..6063,6064..6132))
                     /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     5895..5914
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6133..6237)
                     /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 tggactacaa ggacgatgat gacaagatgg cccccaagaa gaaacggaaa
      961 gtccgcggca tgaagaacaa gggactggca atgactatca ttttccaagc cgaaagcgcc
     1021 aattacggcg aatccctggg gaacatttca agcctgaaga agatctctag gaacaacgga
     1081 gatcagtaca cctacatctc ccgccaagcg atccggtaca atcttatgga tcagatcgga
     1141 gaaaaggaag cccctgtgaa ggctgaaggc ggcggagaca aaaaggtcat ccagttcctg
     1201 tccgaggcca cgatcaccga cttcccggaa ctcgactttt tcggctacct gaaaaccgaa
     1261 aagggttccg ggggacagaa gcgcagcgcc aaagtccgac tgtcgaacgc catctcgctg
     1321 gagactttta agggcgacct ggatttcctc actaacaagg gacaggccga caagctgaac
     1381 gaaaacatga acattgcgca ggcagagatc cacaagtcct actaccgcta caccatcacc
     1441 atcgacctcg accagatcgg tatcgacggg gagattgctc tggacaacaa ggaaaaggcc
     1501 cggagagtga agaagctgat ggacactgtg gcgttcctct atcgggacat taggggcaga
     1561 cgggaggacc tcaagcccct gttcgtgatc ggtggagtgt atgacgtgaa gaacccggtg
     1621 ttccaaaaca ttgtggatgt ggccgacaac aatattgtga tcaagaacat taaggatttg
     1681 ctgacctacg aggacatcaa ggagaacacc agagtcggca tcattgatgg gcagttcgcc
     1741 aactccgacg aagtgaaagt cgagctgaag gccgagtccg tgccagcttt cttcaacgac
     1801 atcaagggaa agattgatgc gtactacgag agcaactgaa gtttaaaccg ctgatcagcc
     1861 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1921 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     1981 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2041 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     2101 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     2161 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2221 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2281 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2341 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2401 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2461 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2521 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     2581 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2641 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     2701 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     2761 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     2821 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     2881 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     2941 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3001 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3061 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3121 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     3181 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3241 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3301 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3361 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3421 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3481 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3541 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3601 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3661 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3721 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     3781 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     3841 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3901 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     3961 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4021 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4081 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4141 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4201 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4261 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4321 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4381 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4441 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4501 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4561 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4621 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4681 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4741 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4801 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4861 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4921 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4981 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5041 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5101 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5161 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5221 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5281 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5341 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5401 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5461 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5521 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5581 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5641 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5701 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5761 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5821 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5881 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5941 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6001 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6061 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6121 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6181 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6241 atttccccga aaagtgccac ctgacgtc
//
