LOCUS       sequence-292292-        8201 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a guide RNA of choice cloned into the BfuI site extended 
            at the 3' end to incorporate a 220-nt control A-tract repeat RNA 
            sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8201)
  AUTHORS   Beltran M, Tavares M, Justin N, Khandelwal G, Ambrose J, Foster BM, 
            Worlock KB, Tvardovskiy A, Kunzelmann S, Herrero J, Bartke T, 
            Gamblin SJ, Wilson JR, Jenner RG
  TITLE     G-tract RNA removes Polycomb repressive complex 2 from genes.
  JOURNAL   Nat Struct Mol Biol. 2019 Oct;26(10):899-909. doi: 
            10.1038/s41594-019-0293-z. Epub 2019 Sep 23.
  PUBMED    31548724
REFERENCE   2  (bases 1 to 8201)
  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"; doi: 
            "10.1038/s41594-019-0293-z"; journalName: "Nat Struct Mol Biol"; 
            date: "2019-10"; volume: "26"; issue: "10"; pages: "899-909"
COMMENT     Sequence Label: pLKO.1-puro U6 sgRNA BfuAI stuffer A-tract
FEATURES             Location/Qualifiers
     source          1..8201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..511
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     430..448
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             533..1132
                     /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(533..552)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1029..1049
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1260..1493
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1565..1686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1602..1621)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1656..1675
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1726..1861
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1881..1900)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1882..1900)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1910..1927)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1910..1926)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1919..1941)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2068..2523
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2155..2174)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2365..2386
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2549..2653
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2654..2722,2723..3514)
                     /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(2872..2891)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3685..4273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4174..4193
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4427..4444
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4561..4582
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  4607..5374
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(5375..5379,5380..5397,5398..5404)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5412..5428
                     /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     5417..5439
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5436..5452
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5436..5452
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5471..5491
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5473..5491
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5519..5745
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5746..5926
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5973..6098
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6591..6824
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7009..7053
                     /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        7351..7599
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7351..7371
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7522..7541
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7633..7708
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    8036..8153
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc
       61 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc
      121 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc
      181 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac
      241 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc
      301 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag
      361 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct
      421 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct
      481 cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga
      541 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
      601 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
      661 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
      721 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
      781 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
      841 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
      901 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
      961 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     1021 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     1081 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc
     1141 ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcgg tacctttaag
     1201 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     1261 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct
     1321 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     1381 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     1441 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag
     1501 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga
     1561 gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1621 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1681 atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact
     1741 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     1801 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     1861 ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     1921 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     1981 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2041 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     2101 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2161 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2221 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2281 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2341 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2401 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2461 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2521 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2581 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2641 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2701 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2761 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2821 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2881 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2941 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3001 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3061 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3121 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3181 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3241 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3301 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3361 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3421 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3481 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3541 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3601 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3661 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3721 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3781 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3841 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3901 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3961 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4021 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4081 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4141 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4201 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4261 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4321 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4381 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4441 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     4501 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     4561 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttggta atgactccaa
     4621 cttattgata gtgttttatg ttcagataat gcccgatgac tttgtcatgc agctccaccg
     4681 attttgagaa cgacagcgac ttccgtccca gccgtgccag gtgctgcctc agattcaggt
     4741 tatgccgctc aattcgctgc gtatatcgct tgctgattac gtgcagcttt cccttcaggc
     4801 gggattcata cagcggccag ccatccgtca tccatatcac cacgtcaaag ggtgacagca
     4861 ggctcataag acgccccagc gtcgccatag tgcgttcacc gaatacgtgc gcaacaaccg
     4921 tcttccggag actgtcatac gcgtaaaaca gccagcgctg gcgcgattta gccccgacat
     4981 agccccactg ttcgtccatt tccgcgcaga cgatgacgtc actgcccggc tgtatgcgcg
     5041 aggttaccga ctgcggcctg agttttttaa gtgacgtaaa atcgtgttga ggccaacgcc
     5101 cataatgcgg gctgttgccc ggcatccaac gccattcatg gccatatcaa tgattttctg
     5161 gtgcgtaccg ggttgagaag cggtgtaagt gaactgcagt tgccatgttt tacggcagtg
     5221 agagcagaga tagcgctgat gtccggcggt gcttttgccg ttacgcacca ccccgtcagt
     5281 agctgaacag gagggacagc tgatagaaac agaagccact ggagcacctc aaaaacacca
     5341 tcatacacta aatcagtaag ttggcagcat caccttacac tttatgcttc cggctcgtat
     5401 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5461 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg
     5521 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     5581 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     5641 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
     5701 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
     5761 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     5821 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     5881 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     5941 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     6001 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     6061 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     6121 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     6181 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     6241 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     6301 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     6361 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     6421 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     6481 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     6541 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     6601 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     6661 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     6721 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     6781 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     6841 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     6901 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     6961 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     7021 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     7081 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     7141 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     7201 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     7261 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgatcacga
     7321 gactagcctc gagcggccgc ccccttcacc gagggcctat ttcccatgat tccttcatat
     7381 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     7441 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     7501 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     7561 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gtgtgcaggt gagtgatcaa
     7621 gaacctgctg acgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     7681 acttgaaaaa gtggcaccga gtcggtgctt cctatgaatc ttaaaaaatg caaaaatctt
     7741 aaaaatgcaa accgtcaaaa tcctacgttc tgcacaaaat gtgccaaaaa tcgtaaaaag
     7801 taaaatggaa aaattctcgg cctgaaaaac tcaaatcaaa aatccaaaaa ctaaaaattc
     7861 tcgggctcta aaatggtaaa aatcgtaaaa agtgaaaatg gaaaaatgtc tgggctcttg
     7921 aaaaactcaa atcaaaaata aaaatttttt tcttaattaa gtctagagtc gactgtttaa
     7981 acctgcaggc atgcgaattc tcgacctcga gacaaatggc agtattcatc cacaatttta
     8041 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     8101 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     8161 attacaggga cagcagagat ccactttggc cgcggctcga g
//
