LOCUS       sequence-90031-e        7288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral Vector for Sp sgRNA delivery without SpCas9, Puromycin 
            resistance, EFS Promoter driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7288)
  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 7288)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951."
COMMENT     Sequence Label: pLKO5.sgRNA.EFS.PAC
FEATURES             Location/Qualifiers
     source          1..7288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..1966
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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"
     CDS             2531..3130
                     /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(2531..2550)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3027..3047
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3146..3734
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3199..3219)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3617..3628)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3806..4039
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4117..4238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4154..4173)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4208..4227
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4278..4413
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4433..4452)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4434..4452)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4462..4479)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4462..4478)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4471..4493)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4620..5075
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4707..4726)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4917..4938
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5101..5205
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5206..5274,5275..6066)
                     /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(5424..5443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6237..6825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6726..6745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6979..6996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7113..7134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7149..7154,7155..7172,7173..7179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7187..7203
                     /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     7192..7214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7211..7227
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7211..7227
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7246..7266
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7248..7266
                     /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 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     2581 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     2641 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     2701 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     2761 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     2821 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     2881 gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa
     2941 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     3001 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     3061 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     3121 cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt
     3181 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3241 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3301 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3361 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3421 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     3481 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3541 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     3601 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3661 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3721 ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc
     3781 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     3841 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     3901 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     3961 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     4021 gtgtggaaaa tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta
     4081 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     4141 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4201 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     4261 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4321 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     4381 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     4441 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     4501 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     4561 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     4621 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4681 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4741 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4801 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4861 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4921 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4981 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5041 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     5101 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5161 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5221 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5281 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5341 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5401 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5461 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5521 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5581 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5641 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5701 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5761 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5821 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5881 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5941 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6001 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6061 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6121 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6181 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6241 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6301 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6361 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6421 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6481 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6541 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6601 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6661 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6721 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6781 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6841 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6901 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6961 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     7021 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     7081 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     7141 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     7201 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     7261 taaagggaac aaaagctgga gctgcaag
//
