LOCUS       sequence-377035-        6950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of puromycin resistance gene and S. aureus 
            scrambled non-targeting gRNA ; useful for changing gRNA by 
            mutagenesis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6950)
  AUTHORS   Sapozhnikov DM, Szyf M
  TITLE     The PROTECTOR strategy employs dCas orthologs to sterically shield 
            off-target sites from CRISPR/Cas activity.
  JOURNAL   Sci Rep. 2023 Feb 9;13(1):2280. doi: 10.1038/s41598-023-29332-2.
  PUBMED    36759683
REFERENCE   2  (bases 1 to 6950)
  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: "Sci Rep."; 
            date: "2023-02-9"; volume: "13(1):2280. doi"; pages: " 
            10.1038/s41598-023-29332-2"
COMMENT     Sequence Label: pLenti-Sa-gRNA-puro
FEATURES             Location/Qualifiers
     source          1..6950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..229
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             230..410
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    457..582
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1075..1308
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1493..1537
                     /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        1893..2141
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1893..1913
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2064..2083
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2162..2237
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     protein_bind    complement(2249..2273)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2346..2387
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2397..2414
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2437..2454)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(2466..2486)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2471..2800
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2651..2786
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2810..3409
                     /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(2810..2829)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3306..3326
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3481..3714
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3786..3907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3823..3842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3877..3896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3947..4082
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4102..4121)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4103..4121)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4131..4148)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4131..4147)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4140..4162)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4289..4744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4376..4395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4586..4607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4770..4874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4875..4943,4944..5735)
                     /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(5093..5112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5906..6494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6395..6414
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6648..6665
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6782..6803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    6846..6862
                     /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     6851..6873
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6870..6886
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6870..6886
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6905..6925
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6907..6925
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
       61 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      121 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      181 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      241 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      301 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      361 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      421 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      481 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      541 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      601 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      661 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
      721 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
      781 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
      841 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
      901 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
      961 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     1021 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     1081 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     1141 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     1201 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     1261 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     1321 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     1381 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggat
     1801 ccagtgtggt ggaattcgcc ctttgtacaa aaaagcaggc tttaaaggaa ccaattcagt
     1861 cgactggatc cggtaccaag gtcgggcagg aagagggcct atttcccatg attccttcat
     1921 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     1981 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2041 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2101 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgcactacca gagctaactc
     2161 agttttagta ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg
     2221 tcaacttgtt ggcgagattt ttttctagac ccagctttct tgtacaaagt tggcattaaa
     2281 gggcgaattc tgcagatatc cagcacagtg gcggccgctc gagtctagag ggcccgcggt
     2341 tcgaaggtaa gcctatccct aaccctctcc tcggtctcga ttctacgcgt accggtcatc
     2401 atcaccatca ccattgagtt taaacctgct gatcagcctc gactgtgcct tctagtcgac
     2461 cctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2521 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2581 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2641 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2701 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2761 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagcttacca tgaccgagta
     2821 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     2881 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     2941 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     3001 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     3061 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     3121 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     3181 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     3241 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     3301 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     3361 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag gtacctttaa
     3421 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     3481 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     3541 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     3601 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     3661 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta
     3721 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     3781 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3841 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3901 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     3961 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4021 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4081 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     4141 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4201 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4261 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     4321 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4381 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4441 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4501 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4561 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4621 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4681 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     4741 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4801 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4861 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4921 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4981 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5041 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5101 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5161 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5221 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5281 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5341 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5401 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5461 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5521 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5581 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5641 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5701 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5761 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5821 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5881 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5941 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6001 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6061 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6121 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6181 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6241 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6301 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6361 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6421 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6481 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6541 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6601 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6661 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6721 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6781 ttaatgtgag ttagctcact cattaggcat acactttatg cttccggctc gtatgttgtg
     6841 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     6901 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt
//
