LOCUS       sequence-398173-        8762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sa-dCas9-NLS-3xFLAG in mammalian cells for enChIP analysis
            to purify specific genomic regions of interest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8762)
  AUTHORS   Fujita T, Yuno M, Fujii H
  TITLE     enChIP systems using different CRISPR orthologues and epitope tags.
  JOURNAL   BMC Res Notes. 2018 Feb 27;11(1):154. doi: 
            10.1186/s13104-018-3262-4.
  PUBMED    29482606
REFERENCE   2  (bases 1 to 8762)
  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: "BMC Res 
            Notes."; date: "2018-02-27"; volume: "11(1):154. doi"; pages: " 
            10.1186/s13104-018-3262-4"
COMMENT     Sequence Label: Sa-dCas9-NLS-3xFLAG/pcDNA3.1
FEATURES             Location/Qualifiers
     source          1..8762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3835..3855
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3862..3927
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4013..4042
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4058..4075
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4119..4136)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4125..4349
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4395..4823
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4482..4501)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4692..4713
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4832..4852)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4837..5166
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5017..5152
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5079..5098
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5233..6027
                     /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(5287..5306)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5897..5916
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6201..6322
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6238..6257)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6292..6311
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6371..6387)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6371..6387)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6384..6406)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6395..6411
                     /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(6419..6425,6426..6443,6444..6449))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6464..6485
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6602..6619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6773..7361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6853..6872)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7532..8323,8324..8392))
                     /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     8155..8174
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8393..8497)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8572..8591)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcatgg gcaaacggaa ctacatcctg gggcttgcca ttgggataac cagcgttggc
      721 tacggaatta ttgattatga gacacgcgat gtgattgacg ccggggttag gctgttcaaa
      781 gaggccaacg ttgaaaacaa cgagggaaga cggagtaagc gcggagcaag aagactcaag
      841 cgcagacgga gacatcggat tcagagggtg aaaaagctgc tcttcgatta caatctcctg
      901 accgatcata gtgagctgag cggaatcaac ccctacgagg cgcgagtgaa agggctttcc
      961 cagaagctgt ccgaagagga gttctccgcc gcgttgctgc acctggccaa acggaggggg
     1021 gttcacaatg taaacgaagt ggaggaggac acgggcaatg aacttagtac gaaagaacag
     1081 atcagtagga actctaaggc tctcgaagag aaatacgtcg ctgagttgca gcttgagaga
     1141 ctgaaaaaag acggcgaagt acgcggatct attaataggt tcaagacttc agattacgta
     1201 aaggaagcca agcagctcct gaaagtacag aaagcgtacc atcagctcga tcagagcttc
     1261 atcgatacct acatagattt gctggagaca cggaggacat actacgaggg cccaggggaa
     1321 ggatctcctt ttgggtggaa ggacatcaag gaatggtacg agatgcttat gggacattgt
     1381 acatattttc cggaggagct caggagcgtc aagtacgcct acaatgccga cctgtacaat
     1441 gccctcaatg acctcaataa cctcgtgatt accagggacg agaacgagaa gctggagtac
     1501 tatgaaaagt tccagattat cgagaatgtg tttaagcaga agaagaagcc gacacttaag
     1561 cagattgcaa aggaaatcct cgtgaatgag gaagatatca agggatacag agtgacaagt
     1621 acaggcaagc ccgagttcac aaatctgaag gtgtaccacg atattaagga cataaccgca
     1681 cgaaaggaga taatcgaaaa cgctgagctc ctcgatcaga tcgcaaaaat tcttaccatc
     1741 taccagtcta gtgaggacat tcaggaggaa ctgactaatc tgaacagtga gctcacccaa
     1801 gaggaaattg agcagatttc aaacctgaaa ggctacaccg ggacgcacaa tctgagcctc
     1861 aaagcaatca acctcattct ggatgaactt tggcacacaa atgacaacca aattgccata
     1921 ttcaaccgcc tgaaactggt gccaaaaaaa gtggatctgt cacagcaaaa ggaaatccct
     1981 acaaccttgg ttgacgattt tattctgtcc cccgttgtca agcggagctt catccagtca
     2041 atcaaggtga tcaatgccat cattaaaaaa tacggattgc caaacgatat aattatcgag
     2101 cttgcacgag agaagaactc aaaggacgcc cagaagatga ttaacgaaat gcagaagcgc
     2161 aaccgccaga caaacgaacg catagaggaa attataagaa caaccggcaa agagaatgcc
     2221 aagtatctga tcgagaaaat caagctgcac gacatgcaag aaggcaagtg cctgtactct
     2281 ctggaagcta tcccactcga agatctgctg aataatccat tcaattacga ggtggacgcc
     2341 atcatcccta gatccgtaag ctttgacaat tccttcaata acaaagttct ggttaaacag
     2401 gaggaaaatt ctaaaaaagg gaaccggacc ccgttccagt acctgagctc cagtgacagc
     2461 aagattagct acgagacttt taagaaacat attctgaatc tggccaaagg caaaggcagg
     2521 atcagcaaga ccaagaagga gtacctcctc gaagaacgcg acattaacag atttagtgtg
     2581 cagaaagatt tcatcaaccg aaaccttgtc gatactcggt acgccacgag aggcctgatg
     2641 aatctcctca ggagctactt ccgcgtcaat aatctggacg ttaaagtcaa gagcataaat
     2701 gggggattca ccagctttct gaggagaaag tggaagttta agaaggaacg aaacaaagga
     2761 tacaagcacc atgctgagga tgctttgatc atcgctaacg cggactttat ctttaaggaa
     2821 tggaaaaagc tggataaggc aaagaaagtg atggaaaacc agatgttcga ggagaagcag
     2881 gcagagtcaa tgcctgagat cgagacagag caggaataca aggaaatttt catcacccct
     2941 catcagatta aacacataaa ggacttcaaa gactataaat actctcatag ggtggacaaa
     3001 aaacccaatc gcgagctcat taatgacacc ctgtactcaa cacggaagga tgataaaggt
     3061 aataccttga ttgtgaataa tcttaatgga ttgtatgaca aagataacga caagctcaag
     3121 aagctgatca acaagtctcc agagaagctc cttatgtatc accacgaccc acagacttat
     3181 cagaaattga aactgatcat ggagcaatac ggggatgaga agaacccact ctacaaatat
     3241 tatgaggaaa caggtaatta cctgaccaag tactccaaga aggataacgg accagtgatc
     3301 aaaaagataa agtactatgg caacaaactt aatgcgcatt tggacataac tgacgattac
     3361 cccaattctc gaaacaaggt tgtgaagctc tccctgaagc cttatagatt tgacgtgtac
     3421 ctggataatg gggtttataa attcgtcacc gtgaaaaatc tggacgtgat caaaaaggag
     3481 aactattatg aagtaaactc aaagtgctat gaggaggcga agaagctgaa gaagatctcc
     3541 aatcaggccg agttcatcgc ttccttctat aataacgatc tcatcaagat caatggagag
     3601 ctttatcgcg tcattggtgt gaacaatgac ttgctgaaca ggatcgaagt caatatgata
     3661 gacattacct accgggagta tctcgaaaac atgaatgata aacggccgcc tcgcatcatc
     3721 aagacaatcg catctaaaac tcagtcaata aaaaagtact ctaccgatat cctggggaat
     3781 ctctatgaag tgaagtcaaa gaagcaccca caaatcatta aaaaaggtgg atcccccaag
     3841 aagaagagga aagtctcgag cgactacaaa gaccatgacg gtgattataa agatcatgac
     3901 atcgattaca aggatgacga tgacaagtaa agcggccgcc actgtgctgg atatctgcag
     3961 aattccacca cactggacta gtggatccga gctcggtacc aagcttgggc ccgaacaaaa
     4021 actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt
     4081 ttaaacggtc tccagcttaa gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg
     4141 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4201 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4261 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     4321 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     4381 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4441 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4501 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4561 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4621 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4681 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4741 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4801 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     4861 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4921 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4981 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5041 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     5101 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     5161 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     5221 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     5281 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     5341 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     5401 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     5461 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     5521 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     5581 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     5641 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     5701 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     5761 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     5821 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     5881 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5941 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     6001 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     6061 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     6121 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     6181 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     6241 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     6301 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     6361 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     6421 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     6481 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6541 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     6601 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6661 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6721 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6781 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6841 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6901 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6961 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     7021 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     7081 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     7141 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     7201 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     7261 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     7321 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     7381 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7441 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7501 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7561 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7621 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7681 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7741 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7801 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7861 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7921 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7981 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     8041 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     8101 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     8161 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     8221 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     8281 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     8341 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     8401 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     8461 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8521 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     8581 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     8641 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     8701 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     8761 tt
//
