LOCUS       Exported                8323 bp ds-DNA     circular SYN 11-JUL-2019
DEFINITION  Expresses S. pyogenes CRISPR chimeric RNA element with customizable 
            sgRNA from U6 promoter and puromycin resistance from EF-1a promoter.
            3rd generation lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    lentiGuide-Puro.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8323)
  AUTHORS   Sanjana NE, Shalem O, Zhang F
  TITLE     Improved vectors and genome-wide libraries for CRISPR screening.
  JOURNAL   Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.
  PUBMED    25075903
REFERENCE   2  (bases 1 to 8323)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Nov 6, 2019 from SnapGene 5.0.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8323
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        162..410
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     162..182
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     277..309
                     /label=AVA1087
     primer_bind     333..352
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     407..431
                     /label=AVA1090
     primer_bind     complement(411..435)
                     /label=AVA1091
     misc_feature    411..431
                     /label=protospacer
     misc_RNA        432..507
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    563..680
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     primer_bind     complement(769..791)
                     /label=AVA1056
     promoter        819..1997
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     intron          1050..1988
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human
                     EF-1-alpha"
     primer_bind     1945..1965
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward
                     primer"
     CDS             2010..2609
                     /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(2010..2029)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also
                     called puro-variant-R"
     primer_bind     complement(2460..2470)
                     /label=AVA1091
     primer_bind     2506..2526
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2625..3213
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     primer_bind     complement(2678..2698)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3096..3107)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3285..3518
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR)
                     from HIV-1"
     polyA_signal    3596..3717
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3633..3652)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3687..3706
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3757..3892
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3819..3838
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3912..3931)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3913..3931)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3941..3958)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
     primer_bind     complement(3941..3957)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3950..3972)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4099..4554
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4186..4205)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4396..4417
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4580..4684
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4685..5545
                     /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     complement(4903..4922)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5716..6304
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6205..6224
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6458..6475
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6592..6613
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        6628..6658
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6666..6682
                     /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)."
     primer_bind     6671..6693
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6690..6706
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6690..6706
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6725..6745
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6727..6745
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6773..6999
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7000..7180
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7227..7352
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     primer_bind     7257..7267
                     /label=AVA1091
     misc_feature    7845..8078
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to 
                     thecytoplasm."
ORIGIN
        1 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
       61 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
      121 tatcgtttca gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga
      181 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
      241 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      301 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      361 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gccgcgcgcg
      421 cgaaaaagcc ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      481 cttgaaaaag tggcaccgag tcggtgcttt tttaagcttg gcgtaactag atcttgagac
      541 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag
      601 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
      661 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc
      721 ggctcgaggg ggcccgggtg caaagatgga taaagtttta aacagagagg aatctttgca
      781 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
      841 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg
      901 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
      961 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     1021 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
     1081 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
     1141 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
     1201 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     1261 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     1321 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     1381 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     1441 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     1501 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     1561 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     1621 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     1681 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     1741 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     1801 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     1861 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     1921 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     1981 catttcaggt gtcgtgacgt acggccacca tgaccgagta caagcccacg gtgcgcctcg
     2041 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     2101 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     2161 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     2221 ccgccgtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     2281 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     2341 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct
     2401 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     2461 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     2521 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     2581 ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt
     2641 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     2701 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     2761 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     2821 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     2881 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     2941 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3001 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3061 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3121 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3181 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa
     3241 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     3301 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     3361 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     3421 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     3481 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca
     3541 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     3601 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3661 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     3721 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3781 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3841 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     3901 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     3961 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4021 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4081 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4141 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4201 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4261 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4321 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4381 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4441 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4501 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     4561 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4621 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4681 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4741 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4801 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4861 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4921 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4981 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5041 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5101 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5161 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5221 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5281 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5341 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5401 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5461 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5521 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5581 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5641 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5701 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5761 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5821 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5881 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5941 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6001 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6061 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6121 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6181 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6241 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6301 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6361 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     6421 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     6481 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     6541 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     6601 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     6661 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     6721 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct
     6781 tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa
     6841 ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta
     6901 ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc
     6961 agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag
     7021 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     7081 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     7141 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
     7201 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
     7261 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
     7321 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     7381 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     7441 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     7501 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     7561 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     7621 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     7681 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     7741 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     7801 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     7861 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     7921 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     7981 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     8041 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     8101 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     8161 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     8221 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     8281 ttggaattag ataaatgggc aagtttgtgg aattggttta aca
//
