LOCUS       Exported                7408 bp ds-DNA     circular SYN 19-JAN-2022
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 Jan 19, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7408
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(13..30)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(264..283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(943..1803)
                     /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     1566..1585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1804..1908)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1934..2389)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2071..2092)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2283..2302
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2516..2538
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2530..2547
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2531..2547
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2557..2576
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2557..2575
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2596..2731
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2650..2669)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2771..2892
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2782..2801)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2836..2855
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             2970..3203
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3275..3863
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3381..3392
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3790..3810
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3885..4598)
                     /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"
     regulatory      4595..4604
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(4624..4835)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    4904..5021
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(5069..5144)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5171..5411)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(5221..5240)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5391..5411)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(5589..5633)
                     /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    5818..6051
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6544..6669
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6716..6896
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(6897..7123)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(7151..7171)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7151..7169)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7190..7206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7190..7206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7203..7225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7214..7230
                     /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(7238..7268)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7283..7304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1921 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     1981 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2041 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2101 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2161 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2221 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2281 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2341 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     2401 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     2461 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     2521 tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa
     2581 ttgggtacgt ccctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
     2641 ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat
     2701 gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg atagctagag
     2761 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     2821 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     2881 aataaacaag ttcctctcac tctctgatat tcatttcttt gcaagttata aatactgaat
     2941 aataagatga catgaactac tatacgtact gctagagatt ttccacactg actaaaaggg
     3001 tctgagggat ctctagttac cagagtcaca caacagacgg gcacacacta cttgaagcac
     3061 tcaaggcaag ctttattgag gcttaagcag tgggttccct agttagccag agagctccca
     3121 ggctcagatc tggtctaacc agagagaccc agtacaagca aaaagcagat cttgtcttcg
     3181 ttgggagtga attagccctt ccagtccccc cttttctttt aaaaagtggc taagatctac
     3241 agctgccttg taagtcattg gtcttaaagt cgacgcgggg aggcggccca aagggagatc
     3301 cgactcgtct gagggcgaag gcgaagacgc ggaagaggcc gcagagccgg cagcaggccg
     3361 cgggaaggaa ggtccgctgg attgagggcc gaagggacgt agcagaagga cgtcccgcgc
     3421 agaatccagg tggcaacaca ggcgagcagc caaggaaagg acgatgattt ccccgacaac
     3481 accacggaat tgtcagtgcc caacagccga gcccctgtcc agcagcgggc aaggcaggcg
     3541 gcgatgagtt ccgccgtggc aatagggagg gggaaagcga aagtcccgga aaggagctga
     3601 caggtggtgg caatgcccca accagtgggg gttgcgtcag caaacacagt gcacaccacg
     3661 ccacgttgcc tgacaacggg ccacaactcc tcataaagag acagcaacca ggatttatac
     3721 aaggaggaga aaatgaaagc catacgggaa gcaatagcat gatacaaagg cattaaagca
     3781 gcgtatccac atagcgtaaa aggagcaaca tagttaagaa taccagtcaa tctttcacaa
     3841 attttgtaat ccagaggttg attgtcgact taacgcgtgg tacctcaatt aagtttgtgc
     3901 cccagtttgc tagggaggtc gcagtatctg gccacagcca cctcgtgctg ctcgacgtag
     3961 gtctctttgt cggcctcctt gattctttcc agtctgtggt ccacatagta gacgccgggc
     4021 atcttgaggt tcttagcggg tttcttggat ctgtatgtgg tcttgaagtt gcagatcagg
     4081 tggcccccgc ccacgagctt cagggccatg tcgcttctgc cttccaggcc gccgtcagcg
     4141 gggtacagca tctcggtgtt ggcctcccag ccgagtgttt tcttctgcat cacagggccg
     4201 ttggatggga agttcacccc tctgatcttg acgttgtaga tgaggcagcc gtcctggagg
     4261 ctggtgtcct gggtagcggt cagcacgccc ccgtcttcgt atgtggtgac tctctcccat
     4321 gtgaagccct cagggaagga ctgcttaaag aagtcgggga tgccctgggt gtggttgatg
     4381 aaggttctgc tgccgtacat gaagctggta gccaggatgt cgaaggcgaa ggggagaggg
     4441 ccgccctcga ccaccttgat tctcatggtc tgggtgccct cgtagggctt gccttcgccc
     4501 tcggatgtgc acttgaagtg gtggttgttc acggtgccct ccatgtacag cttcatgtgc
     4561 atgttctcct taatcagctc ttcgccctta gacaccatgg tggcagatcc cgcgtcacga
     4621 cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     4681 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     4741 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     4801 cttctcgggg actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agccactcga
     4861 gccggcgcca aagtggatct ctgctgtccc tgtaataaac ccgaaaattt tgaatttttg
     4921 taatttgttt ttgtaattct ttagtttgta tgtctgttgc tattatgtct actattcttt
     4981 cccctgcact gtacccccca atcccccctt ttcttttaaa attgtggatg aatactgcca
     5041 tttgtctcaa gatctagaat tcaaaaaagc accgactcgg tgccactttt tcaagttgat
     5101 aacggactag ccttatttta acttgctatt tctagctcta aaacagagac ggtcacgtct
     5161 ccggtgtttc gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt
     5221 acggtaagca tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga
     5281 aattattact ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta
     5341 attctaatta tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga
     5401 aataggccct ctctgggtcc cctcggggtt gggaggtggg tctgaaacga taatggtgaa
     5461 tatccctgcc taactctatt cactatagaa agtacagcaa aaactattct taaacctacc
     5521 aagcctccta ctatcattat gaataatttt atataccaca gccaatttgt tatgttaaac
     5581 caattccaca aacttgccca tttatctaat tccaataatt cttgttcatt cttttcttgc
     5641 tggttttgcg attcttcaat taaggagtgt attaagcttg tgtaattgtt aatttctctg
     5701 tcccactcca tccaggtcgt gtgattccaa atctgttcca gagatttatt actccaacta
     5761 gcattccaag gcacagcagt ggtgcaaatg agttttccag agcaacccca aatccccagg
     5821 agctgttgat cctttaggta tctttccaca gccaggattc ttgcctggag ctgcttgatg
     5881 ccccagactg tgagttgcaa cagatgctgt tgcgcctcaa tagccctcag caaattgttc
     5941 tgctgctgca ctataccaga caataattgt ctggcctgta ccgtcagcgt cattgacgct
     6001 gcgcccatag tgcttcctgc tgctcccaag aacccaagga acaaagctcc tattcccact
     6061 gctctttttt ctctctgcac cactcttctc tttgccttgg tgggtgctac tcctaatggt
     6121 tcaattttta ctactttata tttatataat tcacttctcc aattgtccct catatctcct
     6181 cctccaggtc tgaagatcag cggccgcttg ctgtgcggtg gtcttacttt tgttttgctc
     6241 ttcctctatc ttgtctaaag cttccttggt gtcttttatc tctatccttt gatgcacaca
     6301 atagagggtt gctactgtat tatataatga tctaagttct tctgatcctg tctgaaggga
     6361 tggttgtagc tgtcccagta tttgtctaca gccttctgat gtttctaaca ggccaggatt
     6421 aactgcgaat cgttctagct ccctgcttgc ccatactata tgttttaatt tatatttttt
     6481 ctttccccct ggccttaacc gaattttttc ccatcgcgat ctaattctcc cccgcttaat
     6541 actgacgctc tcgcacccat ctctctcctt ctagcctccg ctagtcaaaa tttttggcgt
     6601 actcaccagt cgccgcccct cgcctcttgc cgtgcgcgct tcagcaagcc gagtcctgcg
     6661 tcgagagagc tcctctggtt tccctttcgc tttcaagtcc ctgttcgggc gccactgcta
     6721 gagattttcc acactgacta aaagggtctg agggatctct agttaccaga gtcacacaac
     6781 agacgggcac acactacttg aagcactcaa ggcaagcttt attgaggctt aagcagtggg
     6841 ttccctagtt agccagagag ctcccaggct cagatctggt ctaaccagag agacccgttt
     6901 atgtatcgag ctaggcactt aaatacaata tctctgcaat gcggcaattc agtggttcgt
     6961 ccaatccatg tcagacccgt ctgttgcctt cctaataagg cacgatcgta ccaccttact
     7021 tccaccaatc ggcatgcacg gtgctttttc tctccttgta aggcatgttg ctaactcatc
     7081 gttaccatgt tgcaagacta caagagtatt gcataagact acattaagct tgcagctcca
     7141 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     7201 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     7261 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     7321 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     7381 cggggagagg cggtttgcgt attgggcg
//
