LOCUS       Exported                8116 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An empty gRNA expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    pKLV-U6gRNA(BbsI)-PGKpuro2ABFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8116)
  AUTHORS   Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera Mdel C, Yusa K
  TITLE     Genome-wide recessive genetic screening in mammalian cells with a 
            lentiviral CRISPR-guide RNA library.
  JOURNAL   Nat Biotechnol. 2014 Mar;32(3):267-73. doi: 10.1038/nbt.2800. Epub 
            2013 Dec 23.
  PUBMED    24535568
REFERENCE   2  (bases 1 to 8116)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8116
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..504
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             522..702
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    749..874
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1371..1604
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1789..1833
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2131..2248
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2331..2571
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2331..2351
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2502..2521
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2596..2671
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2694..3193
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2729..2752)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3126..3145
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3233..3829
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3233..3252)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3729..3749
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3848..3901
                     /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             3902..4603
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4958..4975)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4964..5188
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5214..5234)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5214..5232)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5253..5269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5253..5269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5266..5288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5277..5293
                     /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(5301..5331)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5346..5367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5484..5501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5655..6243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5735..5754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6414..7274)
                     /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     7037..7056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7275..7379)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7405..7860)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7542..7563)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7754..7773
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7987..8009
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8001..8018
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8002..8018
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8028..8047
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8028..8046
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc
      541 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      601 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      661 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      721 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      781 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      841 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      901 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      961 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     1021 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1081 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1141 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1201 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     1261 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     1321 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     1381 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     1441 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     1501 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     1561 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     1621 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     1681 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     1741 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     1801 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     1861 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     1921 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     1981 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     2041 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     2101 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2161 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     2221 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     2281 ttggttagta ccgggcccta cgcgttactc gagccaaggt cgggcaggaa gagggcctat
     2341 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa
     2401 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     2461 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     2521 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     2581 ggtcttcgag aagacgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     2641 tcaacttgaa aaagtggcac cgagtcggtg ctttttttgg atccaattct accgggtagg
     2701 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     2761 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     2821 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     2881 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     2941 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     3001 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     3061 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     3121 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     3181 tccgggcctt tcgacctgca tccatctaga tctcgagcag ctgaagctta ccatgaccga
     3241 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     3301 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     3361 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     3421 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     3481 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     3541 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     3601 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     3661 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     3721 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     3781 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gcggcgggtc
     3841 cggaggagag ggcagaggaa gtctcctaac atgcggtgac gtggaggaga atcctggccc
     3901 aatgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     3961 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     4021 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     4081 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     4141 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aggacggggg
     4201 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     4261 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     4321 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     4381 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     4441 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     4501 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     4561 atactgcgac ctccctagca aactggggca caagcttaat tgagcggccg ctaggtacct
     4621 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4681 ggactggaag ggctaattca ctcccaaaga agtcaagatc tgctttttgc ctgtactggg
     4741 tctctctggt tagaccagag tctctctggt tagaccagat ctgagcctgg gagctctctg
     4801 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     4861 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     4921 tgtggaaaat ctctagcagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     4981 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     5041 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     5101 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaagt caatagcagg
     5161 catgctgggg atgcggtggg ctctatgggc ggccgttaat taagcttttg ttccctttag
     5221 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5281 tatccgctca caattccaca caacatacga gccgggagca taaagtgtaa agcctggggt
     5341 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5401 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5461 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5521 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5581 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5641 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5701 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5761 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5821 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5881 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5941 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6001 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6061 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6121 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6181 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6241 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6301 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6361 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6421 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6481 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6541 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6601 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6661 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6721 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6781 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6841 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6901 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6961 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7021 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7081 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7141 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7201 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7261 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7321 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7381 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     7441 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     7501 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     7561 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     7621 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     7681 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     7741 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     7801 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     7861 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     7921 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     7981 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
     8041 tatagggcga attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     8101 catatatgga gttccg
//
