LOCUS       sequence-381706-        6728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ClipF, sesRNA in mammalian cells, with smV5 and tTA2 
            as efRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6728)
  AUTHORS   Qian Y, Li J, Zhao S, Matthews EA, Adoff M, Zhong W, An X, Yeo M, 
            Park C, Yang X, Wang BS, Southwell DG, Huang ZJ
  TITLE     Programmable RNA sensing for cell monitoring and manipulation.
  JOURNAL   Nature. 2022 Oct;610(7933):713-721. doi: 10.1038/s41586-022-05280-1.
            Epub 2022 Oct 5.
  PUBMED    36198803
REFERENCE   2  (bases 1 to 6728)
  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/s41586-022-05280-1"; journalName: "Nature"; date: 
            "2022-10"; volume: "610"; issue: "7933"; pages: "713-721"
COMMENT     Sequence Label: pAAV-hSyn-ClipF-sesRNA-smV5-tTA2-W3SL
FEATURES             Location/Qualifiers
     source          1..6728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   46..186
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   46..175
                     /label=AAV2 ITR
     rep_origin      261..716
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(348..367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     558..579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(733..752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     852..874
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(912..930)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        998..1102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1103..1171,1172..1963)
                     /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(1321..1340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2134..2722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2623..2642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2784..2913
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2958..3405
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      3419..3428
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4247..4303
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4307..4348
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4358..4399
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4409..4450
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4958..4999
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5009..5050
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5069..5110
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5120..5161
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5357..5398
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5408..5449
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5459..5500
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5513..5566
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(5576..6190,6191..6196,6197..6232,6233..6235,6236..6271,
                     6272..6274,6275..6310,6311..6319)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPAD
                     ALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(6394..6414)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 tttcaggttc agggggagat gtgggaggtt ttttaaagcg gccgcaggaa cccctagtga
       61 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
      121 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
      181 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      241 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
      301 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
      361 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
      421 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
      481 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
      541 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
      601 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
      661 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
      721 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
      781 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
      841 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
      901 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
      961 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1021 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1081 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     1141 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     1201 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     1261 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     1321 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1381 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1441 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     1501 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     1561 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     1621 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     1681 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     1741 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     1801 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     1861 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1921 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1981 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     2041 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2101 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2161 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2221 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2281 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2341 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2401 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2461 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2521 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2581 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2641 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2701 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2761 ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc
     2821 cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
     2881 agggagtggc caactccatc actaggggtt cctgcggccg cacgcgtgtg tctagactgc
     2941 agagggccct gcgtatgagt gcaagtgggt tttaggacca ggatgaggcg gggtgggggt
     3001 gcctacctga cgaccgaccc cgacccactg gacaagcacc caacccccat tccccaaatt
     3061 gcgcatcccc tatcagagag ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg
     3121 ccagcttcag caccgcggac agtgccttcg cccccgcctg gcggcgcgcg ccaccgccgc
     3181 ctcagcactg aaggcgcgct gacgtcactc gccggtcccc cgcaaactcc ccttcccggc
     3241 caccttggtc gcgtccgcgc cgccgccggc ccagccggac cgcaccacgc gaggcgcgag
     3301 ataggggggc acgggcgcga ccatctgcgc tgcggcgccg gcgactcagc gctgcctcag
     3361 tctgcggtgg gcagcggagg agtcgtgtcg tgcctgagag cgcagtcgag aaggatccgc
     3421 caccatggat aaggattgcg agatgaagcg tacaacattg gattccccgt tgggaaagtt
     3481 ggagttgtcc ggatgcgagc agggattgca ccgcattatc ttcttgggaa agggcacctc
     3541 cgctgctgat gctgtcgagg tccccgctcc ggctgctgtc ttgggaggcc ccgagccctt
     3601 gatccaggct acggcttggt tgaacgctta cttccaccag cccgaggcta ttgaggagtt
     3661 ccccgtcccc gctttgcacc accccgtctt ccagcaggag tccttcacgc gtcaggtctt
     3721 gtggaagttg ctgaaggtcg tgaagttcgg cgaggtgatc tccgagtccc acttggctgc
     3781 cttggtggga aacccggctg ccacggccgc tgtcaacacg gctttggatg gcaaccccgt
     3841 cccgatcttg atcccgtgcc accgtgtggt ccagggagat tccgatgtcg gaccctacct
     3901 gggaggcctg gctgtcaagg agtggttgtt ggctcacgag ggacaccgct tgggaaagcc
     3961 cggcttggga gaattcggta agctctgtgg ggatgtctcc aacatattct tcgacatggt
     4021 gacactagac accagattta gaggtttggg agatggtttg ggctctgagg gtcgcatgtg
     4081 caagtaggtc atcattgctg gaagacgttc gcgttcttta gaaagctgta tttctaactg
     4141 ttgcaccacc tgcatttgca ctcgacacgg agcagtgctt cggtcatcca atgcgtgttc
     4201 attgttaagg tgctttaaaa actgtccaaa atcttcacaa aagcttgcta ctaacttcag
     4261 cctgctgaag caggctggag acgtggagga gaaccctgga cctctgggca aacctattcc
     4321 taaccctttg cttggactgg attcaactca gcaacagggc aagcctatcc ccaatcccct
     4381 tcttgggctg gactcaactc agcaacaggg aaaacccatc ccaaatccac tgttgggtct
     4441 ggattccact gggcagggca attctctgat caaggagaac atgaggatga aagtggttat
     4501 ggaagggtct gtaaatgggc atcagttcaa gtgtacgggg gagggcgaag gcaatcccta
     4561 catggggacc cagaccatga ggattaaagt gattgaaggg ggccctctgc cttttgcgtt
     4621 cgatattctg gctacgtcat ttggcggcgg tagtcgcacg ttcatcaaat accccaaggg
     4681 tatacccgac tttttcaaac agagctttcc tgaaggtttt acctgggagc gagtaacccg
     4741 gtatgaagat ggaggggtag tgacggtgat gcaggatacc agccttgaag atgggtgcct
     4801 cgtctaccac gtgcaagtga gaggcgtcaa ctttcctagc aacggcccag tgatgcagaa
     4861 gaagacaaaa ggctgggaac caaatacaga gatgatgtat cccgccgacg ggggcctcag
     4921 gggatatact cacatggctc tgaaagttga tggcggtggc aaaccaattc ctaaccccct
     4981 gctgggcctt gactctaccc agcaacaagg aaagcctatc ccaaatcctc tccttggtct
     5041 ggacagcacc ggtggccagc agggaggcgg gaaaccaatc ccaaaccctc tgttgggttt
     5101 ggattctacc cagcagcagg gtaaacctat accgaaccct ctcctggggc tggactccac
     5161 agggggtgga catcttagct gctcctttgt gactacctac agatccaaga agacggtagg
     5221 gaatattaag atgcctggca ttcacgcggt cgatcatagg ttggagaggc tggaagaaag
     5281 cgataatgaa atgtttgtgg tacagagaga acacgcagtg gctaagtttg ccgggttggg
     5341 aggaggcgga cagggcggca agcctattcc taatcctctg ctcgggttgg atagcacaca
     5401 gcagcaggga aagcccattc caaaccccct cctgggcctg gatagcaccc agcagcaagg
     5461 caaacccatt cccaaccctc tcttgggtct ggactcaacc tacggcgcgc cagagggcag
     5521 aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctttaa ttaactctag
     5581 actggacaag agcaaagtca taaactctgc tctggaatta ctcaatgaag tcggtatcga
     5641 aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt gagcagccta ccctgtactg
     5701 gcacgtgaag aacaagcggg ccctgctcga tgccctggca atcgagatgc tggacaggca
     5761 tcatacccac ttctgccccc tggaaggcga gtcatggcaa gactttctgc ggaacaacgc
     5821 caagtcattc cgctgtgctc tcctctcaca tcgcgacggg gctaaagtgc atctcggcac
     5881 ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca
     5941 aggcttctcc ctggagaacg cactgtacgc tctgtccgcc gtgggccact ttacactggg
     6001 ctgcgtattg gaggatcagg agcatcaagt agcaaaagag gaaagagaga cacctaccac
     6061 cgattctatg cccccacttc tgagacaagc aattgagctg ttcgaccatc agggagccga
     6121 acctgccttc cttttcggcc tggaactaat catatgtggc ctggaaaaac agctaaagtg
     6181 cgaaagcggc ggaccggccg acgcccttga cgattttgac ttagacatgc tcccagccga
     6241 tgcccttgac gactttgacc ttgatatgct gcctgctgac gctcttgacg attttgacct
     6301 tgacatgctc cccgggtaag ctagcaattc cgctcgagat aatcaacctc tggattacaa
     6361 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     6421 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     6481 cttgtataaa tcctggttag ttcttgccac ggcggaactc atcgccgcct gccttgcccg
     6541 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttta tttgtgaaat
     6601 ttgtgatgct attgctttat ttgtaaccat ctagctttat ttgtgaaatt tgtgatgcta
     6661 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     6721 attttatg
//
