pAAV-EFS-CjCas9-eGFP-HIF1a

LOCUS       pAAV-EFS-CjCas9-eGFP-HIF1a                7647 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of cjCas9 with Hif1a targeting sgRNA .
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-EFS-CjCas9-eGFP-HIF1a
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7647)
  AUTHORS   Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, Song DW, Lee KJ, Jung 
            MH, Kim S, Kim JH, Kim JH, Kim JS
  TITLE     In vivo genome editing with a small Cas9 orthologue derived from 
            Campylobacter jejuni.
  JOURNAL   Nat Commun. 2017 Feb 21;8:14500. doi: 10.1038/ncomms14500.
  PUBMED    28220790
REFERENCE   2  (bases 1 to 7647)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..7647
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        67..307
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     67..87
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     238..257
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        339..409
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     promoter        471..682
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             719..3670
                     /codon_start=1
                     /product="Cas9 endonuclease from the Campylobacter jejuni 
                     Type II CRISPR/Cas system (Kim et al., 2016)"
                     /label=CjCas9
                     /translation="MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLA
                     LPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYEL
                     RFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVG
                     EYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKF
                     EEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNT
                     EGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIK
                     ALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFK
                     ALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKE
                     YRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGL
                     KINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVL
                     VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDR
                     NLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTW
                     GFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKR
                     KFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKAL
                     ELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGE
                     IKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFET
                     LSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK"
     CDS             3680..3700
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3701..3727
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3728..3781
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3788..4507
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3788..4504
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3833..3854)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4094..4113)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4441..4462
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(4535..4553)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4536..4553)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4573..4590)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4579..4803
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4832..4961
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5023..5611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5103..5122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5782..6642)
                     /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     6405..6424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6643..6747)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6815..6833
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6871..6893)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6993..7012
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(7029..7484)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7166..7187)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7378..7397
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   7559..52
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7570..52
                     /label=AAV2 ITR
ORIGIN
        1 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
       61 acgcgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
      121 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      181 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      241 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      301 aaaggacgaa acaccgcatg aggaaatgag agaaatgcgt tttagtccct gaagggacta
      361 aaataaagag tttgcgggac tctgcggggt tacaatcccc taaaaccgct tttttgaatt
      421 cgctagctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg
      481 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat ccggtgccta
      541 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
      601 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
      661 cgggtttgcc gccagaacac aggaccggtt ctagagcgct aagcttggga tccgaaccat
      721 ggccagaatc ctggccttcg acatcggcat cagcagcatc ggctgggcct tcagcgagaa
      781 cgacgagctg aaggactgcg gcgtgcggat cttcaccaag gtggaaaacc ccaagaccgg
      841 cgagagcctg gccctgccca gaaggctggc cagaagcgcc cggaagagac tggccagacg
      901 gaaggcccgg ctgaaccacc tgaagcacct gatcgccaac gagttcaagc tgaactacga
      961 ggactaccag agcttcgacg agtccctggc caaggcctac aagggcagcc tgatcagccc
     1021 ctacgagctg cggttccggg ccctgaacga gctgctgagc aagcaggact tcgccagagt
     1081 gatcctgcac attgccaagc ggagaggcta cgacgacatc aagaacagcg acgacaaaga
     1141 gaagggcgcc atcctgaagg ccatcaagca gaacgaggaa aagctggcca actaccagtc
     1201 cgtgggcgag tacctgtaca aagagtactt ccagaagttc aaagagaaca gcaaagaatt
     1261 caccaacgtg cggaacaaga aagaaagcta cgagcggtgt atcgcccaga gcttcctgaa
     1321 ggatgagctg aagctgatct tcaagaagca gagagagttc ggcttcagct tcagcaagaa
     1381 attcgaggaa gaggtgctga gcgtcgcctt ctacaagaga gccctgaagg acttcagcca
     1441 cctcgtgggc aactgcagct tcttcaccga cgagaagaga gcccccaaga acagccccct
     1501 ggccttcatg ttcgtggccc tgacccggat catcaacctg ctgaacaatc tgaagaacac
     1561 cgagggcatc ctgtacacca aggacgacct gaacgccctg ctgaatgagg tgctgaagaa
     1621 cggcaccctg acctacaagc agaccaagaa gctgctgggc ctgagcgacg actacgagtt
     1681 taagggcgag aagggcacct acttcatcga gttcaagaag tacaaagagt tcatcaaggc
     1741 cctgggcgag cacaacctga gccaggacga tctgaatgag atcgccaagg acatcaccct
     1801 gatcaaggac gagattaagc tgaagaaggc cctggccaaa tacgacctga atcagaacca
     1861 gatcgacagc ctgagcaagc tggaattcaa ggatcacctg aacatcagct tcaaggctct
     1921 gaagctggtc acccccctga tgctggaagg caagaagtac gacgaggcct gcaacgagct
     1981 gaacctgaag gtggccatca acgaggacaa gaaggacttc ctgcccgcct tcaacgaaac
     2041 ctactacaag gacgaagtga ccaaccccgt ggtgctgcgg gccatcaaag aataccggaa
     2101 ggtgctgaat gccctgctca agaaatacgg caaggtgcac aagatcaaca tcgagctggc
     2161 ccgggaagtg ggcaagaacc acagccagcg ggccaagatc gagaaagagc agaacgaaaa
     2221 ctacaaggcc aagaaggacg ctgagctgga atgcgagaag ctgggactga agatcaacag
     2281 caagaacatc ctgaagctgc ggctgttcaa agaacagaaa gagttctgcg cctacagcgg
     2341 cgagaagatc aagatcagcg atctgcagga cgagaagatg ctggaaatcg accacatcta
     2401 cccctacagc cggtccttcg acgacagcta catgaacaag gtgctggtgt tcaccaaaca
     2461 gaaccaggaa aaactgaacc agaccccctt cgaggccttc ggcaacgaca gcgccaagtg
     2521 gcagaaaatc gaggtgctgg ccaagaacct gcccaccaag aaacagaaga gaatcctgga
     2581 caagaattac aaggacaaag agcagaagaa cttcaaggac cggaacctga acgacacccg
     2641 gtatatcgcc cggctggtgc tgaactacac aaaggactac ctggatttcc tgcccctgtc
     2701 cgacgacgag aacaccaagc tgaacgatac ccagaaaggc tccaaggtgc acgtggaagc
     2761 caagagcggc atgctgacca gcgccctgag acacacctgg ggcttcagcg ccaaggatcg
     2821 gaacaaccat ctgcaccacg ccatcgacgc cgtgatcatt gcctacgcca acaacagcat
     2881 cgtgaaggcc ttctccgact tcaagaaaga acaggaaagc aacagcgccg agctgtacgc
     2941 caagaagatc tctgagctgg actacaagaa caagcggaag ttcttcgagc ccttcagcgg
     3001 cttccggcag aaggtgctgg ataagatcga cgagatcttc gtgtccaagc ccgagcggaa
     3061 gaagccctct ggcgccctgc acgaggaaac cttcagaaaa gaggaagagt tctaccagtc
     3121 ctacggcggc aaagaaggcg tgctgaaggc cctcgagctg ggcaagatca gaaaagtgaa
     3181 cggcaagatc gtgaagaacg gggacatgtt ccgggtggac atcttcaagc acaaaaagac
     3241 caacaagttc tacgccgtgc ccatctacac catggacttc gccctgaagg tgctgcccaa
     3301 caaggccgtg gcccggtcca agaagggcga gatcaaggac tggattctga tggacgagaa
     3361 ctacgagttc tgctttagcc tgtacaagga ctccctgatc ctgatccaga ccaaggacat
     3421 gcaggaaccc gagttcgtct actacaacgc cttcaccagc agcaccgtgt ccctgatcgt
     3481 gtctaagcac gacaacaagt tcgagacact gagcaagaac cagaagatcc tgttcaagaa
     3541 cgccaacgag aaagaagtga tcgccaagag catcggcatc cagaatctga aggtgttcga
     3601 gaagtacatc gtgtccgccc tgggagaagt gacaaaggcc gagttccggc agagagagga
     3661 cttcaaaaag tccggacctc caaagaaaaa gagaaaagta tacccctacg acgtgcccga
     3721 ctacgccgag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc
     3781 tgaattcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     3841 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     3901 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     3961 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     4021 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     4081 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     4141 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     4201 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     4261 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     4321 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     4381 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     4441 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     4501 caagtaactc gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag
     4561 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4621 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     4681 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4741 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     4801 tggggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     4861 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     4921 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gacatgtgag caaaaggcca
     4981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5041 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5101 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5161 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5221 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5461 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5581 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5701 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5761 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5821 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5881 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5941 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6001 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6061 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6121 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6181 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6241 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6301 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6361 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6421 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6481 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6541 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6601 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6661 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6721 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6781 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6841 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     6901 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     6961 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     7021 gcaccataaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     7081 cagctcattt tttaaccaat agaccgaaat cggcaaaatc ccttataaat caaaagaata
     7141 gcccgagata gagttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     7201 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     7261 atcacccaaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     7321 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     7381 gaagaaagcg aaaggagcgg gcgctaaggc gctggcaagt gtagcggtca cgctgcgcgt
     7441 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtactatg gttgctttga
     7501 cgtatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgcccc
     7561 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg
     7621 gcgacctttg gtcgcccggc ctcagtg
//