LOCUS       sequence-354805-        6960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses puromycin resistance gene and scrambled gRNA for stable 
            integration in mammalian cells; useful as control and template for 
            new gRNAs by mutagenesis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6960)
  AUTHORS   Sapozhnikov DM, Szyf M
  TITLE     Unraveling the functional role of DNA demethylation at specific 
            promoters by targeted steric blockage of DNA methyltransferase with 
            CRISPR/dCas9.
  JOURNAL   Nat Commun. 2021 Sep 29;12(1):5711. doi: 10.1038/s41467-021-25991-9.
  PUBMED    34588447
REFERENCE   2  (bases 1 to 6960)
  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 
            Commun."; date: "2021-09-29"; volume: "12(1):5711. doi"; pages: " 
            10.1038/s41467-021-25991-9"
COMMENT     Sequence Label: pLenti-gRNA-puro
FEATURES             Location/Qualifiers
     source          1..6960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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"
     protein_bind    1827..1851
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_RNA        complement(1863..1938)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1959..2207)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2017..2036)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2187..2207)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             2344..2385
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2395..2412
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2435..2452)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(2464..2484)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2469..2798
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2649..2784
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2808..3407
                     /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(2808..2827)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3304..3324
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3479..3712
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3784..3905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3821..3840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3875..3894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3945..4080
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4100..4119)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4101..4119)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4129..4146)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4129..4145)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4138..4160)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4287..4742
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4374..4393)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4584..4605
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4768..4872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4873..4941,4942..5733)
                     /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(5091..5110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5904..6492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6393..6412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6646..6663
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6780..6801
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6816..6821,6822..6839,6840..6846)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6854..6870
                     /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     6859..6881
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6878..6894
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6878..6894
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6913..6933
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6915..6933
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcggatcc
     1801 agtgtggtgg aattcgccct ttaatgccaa ctttgtacaa gaaagctggg tctagaaaaa
     1861 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
     1921 tatttctagc tctaaaactg agttagctct ggtagtgcgg tgtttcgtcc tttccacaag
     1981 atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt
     2041 ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta
     2101 ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc tctaacagcc
     2161 ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcttc ctgcccgacc
     2221 ttggtaccgg atccagtcga ctgaattggt tcctttaaag cctgcttttt tgtacaaagg
     2281 gcgaattctg cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgcggttc
     2341 gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat
     2401 caccatcacc attgagttta aacctgctga tcagcctcga ctgtgccttc tagtcgaccc
     2461 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     2521 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2581 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2641 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2701 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     2761 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttaccatg accgagtaca
     2821 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     2881 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     2941 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     3001 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     3061 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     3121 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     3181 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     3241 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     3301 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     3361 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaggt acctttaaga
     3421 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     3481 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     3541 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     3601 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     3661 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt
     3721 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     3781 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3841 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3901 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     3961 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4021 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4081 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     4141 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4201 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4261 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     4321 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     4381 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4441 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4501 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4561 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4621 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4681 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     4741 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4801 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4861 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4921 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4981 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5041 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5101 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5161 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5221 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5281 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5341 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5401 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5461 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5521 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5581 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5641 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5701 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5761 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5821 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5881 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5941 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6001 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6061 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6121 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6181 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6241 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6301 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6361 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6421 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6481 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6541 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6601 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6661 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6721 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6781 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6841 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     6901 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
//
