LOCUS       sequence-234992-        7324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-Sirius plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7324)
  AUTHORS   Ma H, Tu LC, Naseri A, Chung YC, Grunwald D, Zhang S, Pederson T
  TITLE     CRISPR-Sirius: RNA scaffolds for signal amplification in genome 
            imaging.
  JOURNAL   Nat Methods. 2018 Nov;15(11):928-931. doi: 
            10.1038/s41592-018-0174-0. Epub 2018 Oct 30.
  PUBMED    30377374
REFERENCE   2  (bases 1 to 7324)
  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/s41592-018-0174-0"; journalName: "Nat Methods"; date: 
            "2018-11"; volume: "15"; issue: "11"; pages: "928-931"
COMMENT     Sequence Label: pHAGE-EFS-PCP-GFPnls
FEATURES             Location/Qualifiers
     source          1..7324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2265..2476
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2522..2531
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2933..3646
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             3683..3703
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3714..4302
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3767..3787)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4185..4196)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4377..4610
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(4729..4747)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4815..4919
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4920..4988,4989..5780)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5138..5157)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5951..6539
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6440..6459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6693..6710
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6950..6971
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6986..6991,6992..7009,7010..7016)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7024..7040
                     /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     7029..7051
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7048..7064
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7048..7064
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7114..7133
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat
     2221 gctagctgat gcgggccctt cgagtggctc cggtgcccgt cagtgggcag agcgcacatc
     2281 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2341 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2401 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2461 tgccgccaga acacaggtgt cgtgacgcgg ttccggttaa ggtgaacgcg tgcaggctgg
     2521 cgccaccatg ggttccaaaa ccatcgttct ttcggtcggc gaggctactc gcactctgac
     2581 tgagatccag tccaccgcag accgtcagat cttcgaagag aaggtcgggc ctctggtggg
     2641 tcggctgcgc ctcacggctt cgctccgtca aaacggagcc aagaccgcgt atcgcgtcaa
     2701 cctaaaactg gatcaggcgg acgtcgttga ttccggactt ccgaaagtgc gctacactca
     2761 ggtatggtcg cacgacgtga caatcgttgc gaatagcacc gaggcctcgc gcaaatcgtt
     2821 gtacgatttg accaagtccc tcgtcgcgac ctcgcaggtc gaagatcttg tcgtcaacct
     2881 tgtgccgctg ggccgtggtg gcggagggac tagtggaggt ggatccggaa gtatgcgtaa
     2941 aggcgaagag ctgttcactg gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa
     3001 cggtcataag ttttccgtgc gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac
     3061 gctgaagttc atctgtacta ctggtaaact gccggtacct tggccgactc tggtaacgac
     3121 gctgacttat ggtgttcagt gctttgctcg ttatccggac catatgaagc agcatgactt
     3181 cttcaagtcc gccatgccgg aaggctatgt gcaggaacgc acgatttcct ttaaggatga
     3241 cggcacgtac aaaacgcgtg cggaagtgaa atttgaaggc gataccctgg taaaccgcat
     3301 tgagctgaaa ggcattgact ttaaagaaga cggcaatatc ctgggccata agctggaata
     3361 caattttaac agccacaatg tttacatcac cgccgataaa caaaaaaatg gcattaaagc
     3421 gaattttaaa attcgccaca acgtggagga tggcagcgtg cagctggctg atcactacca
     3481 gcaaaacact ccaatcggtg atggtcctgt tctgctgcca gacaatcact atctgagcac
     3541 gcaaagcgtt ctgtctaaag atccgaacga gaaacgcgat catatggttc tgctggagtt
     3601 cgtaaccgca gcgggcatca cgcatggtat ggatgaactg tacaaatctg gcggccgctc
     3661 tggcatcgat gggagcctcg agccaaagaa aaagcggaaa gtgtaatcta gataatcaac
     3721 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     3781 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     3841 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     3901 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     3961 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     4021 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     4081 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
     4141 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     4201 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     4261 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctgagatc ctttaagacc
     4321 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     4381 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     4441 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     4501 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     4561 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
     4621 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     4681 gcccgggtta attaaggaaa gggctagatc attcttgaag acgaaagggc ctcgtgatac
     4741 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4801 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4861 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4921 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4981 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5041 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5101 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5161 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5221 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5281 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5341 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5401 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5461 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5521 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5581 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5641 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5701 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5761 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5821 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5881 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5941 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6001 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6061 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6121 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6181 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6241 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6301 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6361 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6421 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6481 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6541 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6601 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     6661 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     6721 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcaagc
     6781 tcatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     6841 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctccccgt
     6901 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6961 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     7021 gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt acgaatttca
     7081 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     7141 cttatcatgt ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca
     7201 ccccataccc tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt
     7261 cctctgaatt attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag
     7321 tagt
//
