LOCUS       Exported                7408 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  3rd generation Lentiviral Vector for Sp sgRNA delivery without 
            SpCas9, tagRFP, EFS Promoter driven.
ACCESSION   .
VERSION     .
KEYWORDS    pLKO5.sgRNA.EFS.tRFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7408)
  AUTHORS   Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey 
            ME, Thielke A, Aster JC, Regev A, Ebert BL
  TITLE     Generation of mouse models of myeloid malignancy with combinatorial 
            genetic lesions using CRISPR-Cas9 genome editing.
  JOURNAL   Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
  PUBMED    24952903
REFERENCE   2  (bases 1 to 7408)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7408
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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"
     promoter        1717..1957
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1984..2059
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2107..2224
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2293..2504
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2524..2533
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2530..3243
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    3265..3853
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3318..3338)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3736..3747)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3925..4158
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4236..4357
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4273..4292)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4327..4346
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4397..4532
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4459..4478
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4552..4571)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4553..4571)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4581..4598)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4581..4597)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4590..4612)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4739..5194
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4826..4845)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5036..5057
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5220..5324
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5325..6185
                     /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(5543..5562)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6356..6944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6845..6864
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7098..7115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7232..7253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7268..7298
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7306..7322
                     /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     7311..7333
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7330..7346
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7330..7346
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7365..7385
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7367..7385
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cgtgaccgtc
     1981 tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2041 agtggcaccg agtcggtgct tttttgaatt ctagatcttg agacaaatgg cagtattcat
     2101 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     2161 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     2221 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg
     2281 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     2341 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     2401 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     2461 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga
     2521 tctgccacca tggtgtctaa gggcgaagag ctgattaagg agaacatgca catgaagctg
     2581 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag
     2641 ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc
     2701 gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat caaccacacc
     2761 cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc
     2821 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc
     2881 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg
     2941 cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc tgacggcggc
     3001 ctggaaggca gaagcgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac
     3061 ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
     3121 tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag
     3181 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat
     3241 tgaggtacca cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg
     3301 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     3361 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     3421 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     3481 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     3541 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     3601 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     3661 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     3721 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     3781 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     3841 gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     3901 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     3961 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     4021 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     4081 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     4141 tgtggaaaat ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat
     4201 aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg
     4261 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4321 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc
     4381 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4441 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     4501 ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt
     4561 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4621 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4681 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     4741 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4801 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4861 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4921 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4981 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5041 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     5101 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5161 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     5221 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5281 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5341 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5401 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5461 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5521 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5581 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5641 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5701 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5761 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5821 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5881 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5941 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6001 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6061 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6121 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6181 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6241 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6301 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6361 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6421 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6481 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6541 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6601 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6661 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6721 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6781 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6841 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6901 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6961 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     7021 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     7081 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     7141 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     7201 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     7261 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     7321 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
     7381 aaagggaaca aaagctggag ctgcaagc
//
