LOCUS       sequence-366141-        7310 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA with MS2 aptamer, TagRFP657 as selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7310)
  AUTHORS   Chan WF, Coughlan HD, Chen Y, Keenan CR, Smyth GK, Perkins AC, 
            Johanson TM, Allan RS
  TITLE     Activation of stably silenced genes by recruitment of a synthetic 
            de-methylating module.
  JOURNAL   Nat Commun. 2022 Sep 23;13(1):5582. doi: 10.1038/s41467-022-33181-4.
  PUBMED    36151095
REFERENCE   2  (bases 1 to 7310)
  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: "2022-09-23"; volume: "13(1):5582. doi"; pages: " 
            10.1038/s41467-022-33181-4"
COMMENT     Sequence Label: gRNA-MS2x2-TagRFP657
FEATURES             Location/Qualifiers
     source          1..7310
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        127..145
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        197..215
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    325..442
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        491..1001
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     920..938
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     LTR             1826..2059
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2131..2252
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2168..2187)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2222..2241
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2292..2427
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2447..2466)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2448..2466)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2476..2493)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2476..2492)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2485..2507)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2634..3089
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2721..2740)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2931..2952
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3115..3219
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3220..3288,3289..4080)
                     /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(3438..3457)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4251..4839
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4740..4759
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4993..5010
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5127..5148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5163..5168,5169..5186,5187..5193)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5201..5217
                     /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     5206..5228
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5225..5241
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5225..5241
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5260..5280
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5262..5280
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5308..5534
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5535..5715
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5762..5887
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6380..6613
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6798..6842
                     /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        join(7140..7310,1..78)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7140..7160
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
       61 tggaaaggac gaaacaccgg tgtcttcgag gcttacagga cgaagacccg tttgagagct
      121 aggccaacat gaggatcacc catgtctgca gggcctagca agttcaaata aggctagtcc
      181 gttatcaact tggccaacat gaggatcacc catgtctgca gggccaagtg gcaccgagtc
      241 ggtgcttttt ttgaaatgtc gacctcgaga gtctagaaat actgaattct cgacctcgag
      301 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
      361 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
      421 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcc
      481 gcggctcgag ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac
      541 gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat
      601 tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg
      661 cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga
      721 cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag
      781 caatggcagc gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg
      841 ccgagagcag cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg
      901 ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag
      961 tcggctccct cgttgaccga atcaccgacc tctctcccca gggggatctc tcgaggttgc
     1021 caccatgagc gagctgataa cagaaaacat gcacatgaag ctctacatgg aaggtaccgt
     1081 caacaaccac catttcaagt gtacttctga aggcgaaggt aagccttacg aaggcaccca
     1141 aacccaacgc attaaagtag tggaaggggg acccctgccc ttcgccttcg atattttggc
     1201 aacaagcttc atgtacggaa gtcatacgtt tatcaatcat acgcagggga tccctgattt
     1261 ctggaaacag agtttcccag aggggttcac gtgggagcga gtaactactt acgaggacgg
     1321 cggggtactg acagccacac aagatacatc tcttcaggat ggctgcttga tctataatgt
     1381 gaagattcgc ggcgttaact ttcccagtaa cggccctgtc atgcagaaaa aaacgcttgg
     1441 gtgggaggct cacacagaaa tgctctaccc agcagatggg ggtctggagg gtagaactgc
     1501 tctggccctg aagctcgtgg gcggcggtca cctcatttgc aactttaaaa ccacctatcg
     1561 gtctaagaag ccagcaaaga acctcaagat gcccggagtt tattatgttg actacaggct
     1621 cgagcggatc aaggaagctg acaaggagac ctacgtggag cagcatgaag ttgcagtagc
     1681 tcgctactgc gatttgccat ctaagttggg ccataagctg aattgaaacg aatttcgacc
     1741 tcgagggggg gcccggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     1801 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagaaaagat
     1861 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     1921 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     1981 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     2041 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt
     2101 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     2161 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     2221 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     2281 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2341 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     2401 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     2461 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2521 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2581 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     2641 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2701 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2761 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2821 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2881 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2941 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3001 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3061 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     3121 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3181 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3241 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3301 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3361 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3421 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3481 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3541 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3601 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3661 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3721 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3781 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3841 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3901 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3961 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4021 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4081 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4141 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4201 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4261 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4321 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4381 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4441 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4501 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4561 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4621 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4681 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4741 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4801 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4861 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4921 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4981 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     5041 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     5101 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     5161 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     5221 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
     5281 gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca
     5341 acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc
     5401 gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga
     5461 catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag
     5521 ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct
     5581 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     5641 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     5701 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
     5761 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
     5821 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
     5881 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
     5941 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
     6001 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     6061 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     6121 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     6181 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca
     6241 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     6301 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     6361 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     6421 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     6481 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     6541 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     6601 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     6661 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     6721 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     6781 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     6841 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     6901 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     6961 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     7021 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     7081 tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc cccttcaccg
     7141 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     7201 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     7261 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
//
