LOCUS       sequence-336049-        7100 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of a SunTag-dCjCas9 fusion protein in which 10 copies of 
            the GCN4 peptide are fused to the N terminus of dCjCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7100)
  AUTHORS   Zhang X, Lv S, Luo Z, Hu Y, Peng X, Lv J, Zhao S, Feng J, Huang G, 
            Wan QL, Liu J, Huang H, Luan B, Wang D, Zhao X, Lin Y, Zhou Q, Zhang
            ZN, Rong Z
  TITLE     MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional 
            activator, elicits gene expression in vivo.
  JOURNAL   Nucleic Acids Res. 2021 Mar 22. pii: 6179929. doi: 
            10.1093/nar/gkab174.
  PUBMED    33751124
REFERENCE   2  (bases 1 to 7100)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2021 Mar 22. pii: 6179929. doi: 10.1093/nar/gkab174."
COMMENT     Sequence Label: pRGEN-CMV-10XGCN4-L1-dCjCas9 S-D
FEATURES             Location/Qualifiers
     source          1..7100
                     /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"
     regulatory      663..672
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             672..728
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             744..800
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             816..872
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             888..944
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             960..1016
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1032..1088
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1104..1160
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1176..1232
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1248..1304
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1320..1376
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1410..1430
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1437..1457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1542..4493
                     /codon_start=1
                     /product="Cas9 endonuclease from the Campylobacter jejuni 
                     Type II CRISPR/Cas system (Kim et al., 2016)"
                     /label=CjCas9
                     /translation="MARILAFAIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLA
                     LPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYEL
                     RFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVG
                     EYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKF
                     EEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNT
                     EGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIK
                     ALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFK
                     ALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKE
                     YRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGL
                     KINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDAIYPYSRSFDDSYMNKVL
                     VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDR
                     NLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTW
                     GFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKR
                     KFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKAL
                     ELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGE
                     IKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFET
                     LSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK"
     CDS             4503..4523
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4524..4550
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             4524..4550
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(4584..4602)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4585..4602)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4622..4639)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4628..4852
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4940..4957)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5111..5699)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5191..5210)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5870..6661,6662..6730))
                     /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     6493..6512
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6731..6835)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6910..6929)
                     /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 acccaagctt
      661 gggccaccat ggaagaactt ttgagcaaga attatcatct tgagaacgaa gtggctcgtc
      721 ttaagaaagg ttctggcagt ggagaagaac tgctttcaaa gaattaccac ctggaaaatg
      781 aggtagctag actgaaaaag gggagcggaa gtggggagga gttgctgagc aaaaattatc
      841 atttggagaa cgaagtagca cgactaaaga aagggtccgg atcgggtgag gagttactct
      901 cgaaaaatta tcatctcgaa aacgaagtgg ctcggctaaa aaagggcagt ggttctggag
      961 aagagctatt atctaaaaac taccacctcg aaaatgaggt ggcacgctta aaaaagggaa
     1021 gtggcagtgg tgaagagcta ctatccaaga attatcatct tgagaacgag gtagcgcgtt
     1081 tgaagaaggg ttccggctca ggagaggaac tgctctcgaa gaactatcat cttgaaaatg
     1141 aggtcgctcg attaaaaaag ggatcgggca gtggtgagga actactttca aagaattacc
     1201 acctcgaaaa cgaagtagct cgattaaaga aaggttcagg gtcgggtgaa gaattactga
     1261 gtaaaaatta tcatctggaa aatgaggtag cgagactaaa aaaggggagt ggttctggcg
     1321 aggaattgct atcgaaaaat tatcatcttg agaacgaagt tgctaggctc aaaaagggct
     1381 caggctcagg cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg gaagatccta
     1441 agaaaaagcg gaaagtggac ggcattggta gtgggagcaa cggcagcagc ggatccaacg
     1501 gtccgggtgg atctggaggt ggaggttctg gaggacgaat catggccaga atcctggcct
     1561 tcgctatcgg catcagcagc atcggctggg ccttcagcga gaacgacgag ctgaaggact
     1621 gcggcgtgcg gatcttcacc aaggtggaaa accccaagac cggcgagagc ctggccctgc
     1681 ccagaaggct ggccagaagc gcccggaaga gactggccag acggaaggcc cggctgaacc
     1741 acctgaagca cctgatcgcc aacgagttca agctgaacta cgaggactac cagagcttcg
     1801 acgagtccct ggccaaggcc tacaagggca gcctgatcag cccctacgag ctgcggttcc
     1861 gggccctgaa cgagctgctg agcaagcagg acttcgccag agtgatcctg cacattgcca
     1921 agcggagagg ctacgacgac atcaagaaca gcgacgacaa agagaagggc gccatcctga
     1981 aggccatcaa gcagaacgag gaaaagctgg ccaactacca gtccgtgggc gagtacctgt
     2041 acaaagagta cttccagaag ttcaaagaga acagcaaaga attcaccaac gtgcggaaca
     2101 agaaagaaag ctacgagcgg tgtatcgccc agagcttcct gaaggatgag ctgaagctga
     2161 tcttcaagaa gcagagagag ttcggcttca gcttcagcaa gaaattcgag gaagaggtgc
     2221 tgagcgtcgc cttctacaag agagccctga aggacttcag ccacctcgtg ggcaactgca
     2281 gcttcttcac cgacgagaag agagccccca agaacagccc cctggccttc atgttcgtgg
     2341 ccctgacccg gatcatcaac ctgctgaaca atctgaagaa caccgagggc atcctgtaca
     2401 ccaaggacga cctgaacgcc ctgctgaatg aggtgctgaa gaacggcacc ctgacctaca
     2461 agcagaccaa gaagctgctg ggcctgagcg acgactacga gtttaagggc gagaagggca
     2521 cctacttcat cgagttcaag aagtacaaag agttcatcaa ggccctgggc gagcacaacc
     2581 tgagccagga cgatctgaat gagatcgcca aggacatcac cctgatcaag gacgagatta
     2641 agctgaagaa ggccctggcc aaatacgacc tgaatcagaa ccagatcgac agcctgagca
     2701 agctggaatt caaggatcac ctgaacatca gcttcaaggc tctgaagctg gtcacccccc
     2761 tgatgctgga aggcaagaag tacgacgagg cctgcaacga gctgaacctg aaggtggcca
     2821 tcaacgagga caagaaggac ttcctgcccg ccttcaacga aacctactac aaggacgaag
     2881 tgaccaaccc cgtggtgctg cgggccatca aagaataccg gaaggtgctg aatgccctgc
     2941 tcaagaaata cggcaaggtg cacaagatca acatcgagct ggcccgggaa gtgggcaaga
     3001 accacagcca gcgggccaag atcgagaaag agcagaacga aaactacaag gccaagaagg
     3061 acgctgagct ggaatgcgag aagctgggac tgaagatcaa cagcaagaac atcctgaagc
     3121 tgcggctgtt caaagaacag aaagagttct gcgcctacag cggcgagaag atcaagatca
     3181 gcgatctgca ggacgagaag atgctggaaa tcgacgccat ctacccctac agccggtcct
     3241 tcgacgacag ctacatgaac aaggtgctgg tgttcaccaa acagaaccag gaaaaactga
     3301 accagacccc cttcgaggcc ttcggcaacg acagcgccaa gtggcagaaa atcgaggtgc
     3361 tggccaagaa cctgcccacc aagaaacaga agagaatcct ggacaagaat tacaaggaca
     3421 aagagcagaa gaacttcaag gaccggaacc tgaacgacac ccggtatatc gcccggctgg
     3481 tgctgaacta cacaaaggac tacctggatt tcctgcccct gtccgacgac gagaacacca
     3541 agctgaacga tacccagaaa ggctccaagg tgcacgtgga agccaagagc ggcatgctga
     3601 ccagcgccct gagacacacc tggggcttca gcgccaagga tcggaacaac catctgcacc
     3661 acgccatcga cgccgtgatc attgcctacg ccaacaacag catcgtgaag gccttctccg
     3721 acttcaagaa agaacaggaa agcaacagcg ccgagctgta cgccaagaag atctctgagc
     3781 tggactacaa gaacaagcgg aagttcttcg agcccttcag cggcttccgg cagaaggtgc
     3841 tggataagat cgacgagatc ttcgtgtcca agcccgagcg gaagaagccc tctggcgccc
     3901 tgcacgagga aaccttcaga aaagaggaag agttctacca gtcctacggc ggcaaagaag
     3961 gcgtgctgaa ggccctcgag ctgggcaaga tcagaaaagt gaacggcaag atcgtgaaga
     4021 acggggacat gttccgggtg gacatcttca agcacaaaaa gaccaacaag ttctacgccg
     4081 tgcccatcta cacaatggac ttcgccctga aggtgctgcc caacaaggcc gtggcccggt
     4141 ccaagaaggg cgagatcaag gactggattc tgatggacga gaactacgag ttctgcttta
     4201 gcctgtacaa ggactccctg atcctgatcc agaccaagga catgcaggaa cccgagttcg
     4261 tctactacaa cgccttcacc agcagcaccg tgtccctgat cgtgtctaag cacgacaaca
     4321 agttcgagac actgagcaag aaccagaaga tcctgttcaa gaacgccaac gagaaagaag
     4381 tgatcgccaa gagcatcggc atccagaatc tgaaggtgtt cgagaagtac atcgtgtccg
     4441 ccctgggaga agtgacaaag gccgagttcc ggcagagaga ggacttcaaa aagtccggac
     4501 ctccaaagaa aaagagaaaa gtatacccct acgacgtgcc cgactacgcc taataactcg
     4561 agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca
     4621 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4681 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4741 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4801 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     4861 gcggaaagaa ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4921 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4981 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5041 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5101 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5161 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5221 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5281 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5341 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5401 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5461 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5521 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5581 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5641 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5701 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5761 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5821 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5881 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5941 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6001 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6061 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6121 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6181 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6241 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6301 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6361 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6421 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6481 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6541 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6601 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6661 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6721 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6781 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6841 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     6901 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     6961 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     7021 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     7081 acgggccaga tatacgcgtt
//
