LOCUS       Exported                7408 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  3rd generation lentiviral Vector for Sp sgRNA delivery without 
            SpCas9, GFP, EFS Promoter driven.
ACCESSION   .
VERSION     .
KEYWORDS    pLKO5.sgRNA.EFS.GFP
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        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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        1718..1958
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1718..1738
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1889..1908
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1985..2060
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2108..2225
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2294..2505
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2525..2534
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2531..3250
                     /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             2531..3247
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2576..2597)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2837..2856)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3184..3205
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3266..3854
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3319..3339)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3737..3748)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3926..4159
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4237..4358
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4274..4293)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4328..4347
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4398..4533
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4460..4479
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4553..4572)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4554..4572)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4582..4599)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4582..4598)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4591..4613)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4740..5195
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4827..4846)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5037..5058
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5221..5325
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5326..6186
                     /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(5544..5563)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6357..6945
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6846..6865
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7099..7116
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7233..7254
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7269..7299
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7307..7323
                     /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     7312..7334
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7331..7347
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7331..7347
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7366..7386
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7368..7386
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc
     1741 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     1801 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     1861 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     1921 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag acgtgaccgt
     1981 ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     2041 aagtggcacc gagtcggtgc ttttttgaat tctagatctt gagacaaatg gcagtattca
     2101 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     2161 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     2221 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagtggctcc
     2281 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     2341 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     2401 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     2461 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc gtgacgcggg
     2521 atccgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2581 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     2641 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2701 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2761 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2821 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2881 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2941 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     3001 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3061 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3121 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     3181 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3241 gtacaagtaa acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt
     3301 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3361 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3421 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3481 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3541 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     3601 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3661 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     3721 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3781 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3841 ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc
     3901 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     3961 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     4021 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     4081 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     4141 gtgtggaaaa tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta
     4201 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     4261 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4321 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     4381 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4441 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     4501 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     4561 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     4621 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     4681 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     4741 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4801 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4861 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4921 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4981 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5041 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     5101 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5161 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     5221 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5281 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5341 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5401 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5461 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5521 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5581 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5641 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5701 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5761 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5821 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5881 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5941 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6001 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6061 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6121 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6181 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6241 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6301 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6361 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6421 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6481 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6541 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6601 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6661 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6721 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6781 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6841 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6901 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6961 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     7021 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     7081 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     7141 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     7201 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     7261 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     7321 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     7381 taaagggaac aaaagctgga gctgcaag
//
