LOCUS       sequence-306269-        6255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty SgRNA expression system matching CasRx.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6255)
  AUTHORS   He B, Peng W, Huang J, Zhang H, Zhou Y, Yang X, Liu J, Li Z, Xu C, 
            Xue M, Yang H, Huang P
  TITLE     Modulation of metabolic functions through Cas13d-mediated gene 
            knockdown in liver.
  JOURNAL   Protein Cell. 2020 Mar 18. pii: 10.1007/s13238-020-00700-2. doi: 
            10.1007/s13238-020-00700-2.
  PUBMED    32185621
REFERENCE   2  (bases 1 to 6255)
  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: "Protein 
            Cell. 2020 Mar 18. pii: 10.1007/s13238-020-00700-2. doi: 
            10.1007/s13238-020-00700-2."
COMMENT     Sequence Label: PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40
FEATURES             Location/Qualifiers
     source          1..6255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    105..126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(141..146,147..164,165..171)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    179..195
                     /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     184..206
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     203..219
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     203..219
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   248..282
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        593..841
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     593..613
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     764..783
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        843..878
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        897..932
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        1003..1214
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1227..1495
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1523..1532
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1529..2239
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1674..1692)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1942..1961
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2240..2361
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2277..2296)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2331..2350
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   2539..2601
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2686..2705)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2687..2705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2712..2729)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2712..2728)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2721..2743)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2869..3297
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2956..2975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3166..3187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3641..4435
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3695..3714)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4305..4324
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4453..4521,4522..5313)
                     /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(4671..4690)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5484..6072
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5973..5992
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6226..6243
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
       61 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      121 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      181 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcttggt
      241 accttaaccc tagaaagata gtctgcgtaa aattgacgca tgcattcttg aaatattgct
      301 ctctctttct aaatagcgcg aatccgtcgc tgtgcattta ggacatctca gtcgccgctt
      361 ggagctcccg tgaggcgtgc ttgtcaatgc ggtaagtgtc actgattttg aactataacg
      421 accgcgtgag tcaaaatgac gcatgattat cttttacgtg acttttaaga tttaactcat
      481 acgataatta tattgttatt tcatgttcta cttacgtgat aacttattat atatatattt
      541 tcttgttata gatatcatta atatcgatgg gctggccttt tgctcagcta gcgagggcct
      601 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
      661 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
      721 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
      781 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
      841 cgcaagtaaa cccctaccaa ctggtcgggg tttgaaacgg gtcttcgaga agacctcaag
      901 taaaccccta ccaactggtc ggggtttgaa actttttttg aattctgatg cggtattttc
      961 tccttacgca aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc
     1021 ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt
     1081 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     1141 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     1201 ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc
     1261 ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg
     1321 cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt
     1381 gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc
     1441 tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc
     1501 tgtgaccggc gcctacaccg gtgccaccat ggtgagcaag ggcgaggagg ataacatggc
     1561 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     1621 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     1681 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     1741 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     1801 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     1861 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     1921 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     1981 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     2041 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     2101 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     2161 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     2221 ggacgagctg tacaagtaaa acttgtttat tgcagcttat aatggttaca aataaagcaa
     2281 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2341 caaactcatc aatgtatctt acctaaaagt tttgttactt tatagaagaa attttgagtt
     2401 tttgtttttt tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag
     2461 tatgtaagtg taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat
     2521 atacagaccg ataaaacaca tgcgtcaatt ttacgcatga ttatctttaa cgtacgtcac
     2581 aatatgatta tctttctagg gttaaggccg gccaataagg gcgaattctg cagatatcca
     2641 tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg
     2701 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     2761 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     2821 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta
     2881 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2941 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3001 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3061 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3121 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3181 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3241 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3301 acaaaattca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag ccagtccgca
     3361 gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc
     3421 aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc
     3481 ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggttgg
     3541 gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg
     3601 atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3661 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3721 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3781 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3841 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3901 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catcccacct
     3961 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     4021 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     4081 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     4141 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     4201 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4261 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4321 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4381 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgaattga
     4441 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4501 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4561 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4621 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4681 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4741 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4801 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4861 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4921 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4981 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5041 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5101 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5161 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5221 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5281 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5341 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5401 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5461 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5521 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5581 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5641 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5701 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5761 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5821 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5881 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5941 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6001 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6061 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6121 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6181 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6241 gaggaagcgg aagag
//
