LOCUS       sequence-472762-        6665 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 6665)
  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 6665)
  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-HA-2a-Hu VGAT
            sesRNA-2a-smV5-2...
FEATURES             Location/Qualifiers
     source          1..6665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      94..682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     583..602
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   744..873
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        918..1365
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1379..1388
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2144..2200
                     /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             2204..2245
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2255..2296
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2306..2347
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2855..2896
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2906..2947
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2966..3007
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3017..3058
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3254..3295
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3305..3346
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3356..3397
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3410..3463
                     /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(3473..4087,4088..4093,4094..4129,4130..4132,4133..4168,
                     4169..4171,4172..4207,4208..4216)
                     /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(4291..4311)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4671..4811
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4671..4800
                     /label=AAV2 ITR
     rep_origin      4886..5341
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4973..4992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5183..5204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5358..5377)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5477..5499
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5537..5555)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5623..5727
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5728..5796,5797..6588)
                     /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(5946..5965)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
       61 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
      121 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
      181 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
      241 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      301 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      361 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      421 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      481 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      541 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      601 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      661 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      721 ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc
      781 cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
      841 agggagtggc caactccatc actaggggtt cctgcggccg cacgcgtgtg tctagactgc
      901 agagggccct gcgtatgagt gcaagtgggt tttaggacca ggatgaggcg gggtgggggt
      961 gcctacctga cgaccgaccc cgacccactg gacaagcacc caacccccat tccccaaatt
     1021 gcgcatcccc tatcagagag ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg
     1081 ccagcttcag caccgcggac agtgccttcg cccccgcctg gcggcgcgcg ccaccgccgc
     1141 ctcagcactg aaggcgcgct gacgtcactc gccggtcccc cgcaaactcc ccttcccggc
     1201 caccttggtc gcgtccgcgc cgccgccggc ccagccggac cgcaccacgc gaggcgcgag
     1261 ataggggggc acgggcgcga ccatctgcgc tgcggcgccg gcgactcagc gctgcctcag
     1321 tctgcggtgg gcagcggagg agtcgtgtcg tgcctgagag cgcagtcgag aaggatccgc
     1381 caccatggat aaggattgcg agatgaagcg tacaacattg gattccccgt tgggaaagtt
     1441 ggagttgtcc ggatgcgagc agggattgca ccgcattatc ttcttgggaa agggcacctc
     1501 cgctgctgat gctgtcgagg tccccgctcc ggctgctgtc ttgggaggcc ccgagccctt
     1561 gatccaggct acggcttggt tgaacgctta cttccaccag cccgaggcta ttgaggagtt
     1621 ccccgtcccc gctttgcacc accccgtctt ccagcaggag tccttcacgc gtcaggtctt
     1681 gtggaagttg ctgaaggtcg tgaagttcgg cgaggtgatc tccgagtccc acttggctgc
     1741 cttggtggga aacccggctg ccacggccgc tgtcaacacg gctttggatg gcaaccccgt
     1801 cccgatcttg atcccgtgcc accgtgtggt ccagggagat tccgatgtcg gaccctacct
     1861 gggaggcctg gctgtcaagg agtggttgtt ggctcacgag ggacaccgct tgggaaagcc
     1921 cggcttggga gaattcggac ttcaggcgcc cgtcgccgct gtagcaggcc gggaaaaagg
     1981 cgcggctgcc ttcctggaag agcgacttct ccagcacctc gacagcggca aagaatagca
     2041 gaggatagga caacagcgcc ttggccacca gaaagatgtt gaccacggcg cggatggagc
     2101 cgggcaggtt atccgtgatg acctccttgg tctcgtcaag cttgctacta acttcagcct
     2161 gctgaagcag gctggagacg tggaggagaa ccctggacct ctgggcaaac ctattcctaa
     2221 ccctttgctt ggactggatt caactcagca acagggcaag cctatcccca atccccttct
     2281 tgggctggac tcaactcagc aacagggaaa acccatccca aatccactgt tgggtctgga
     2341 ttccactggg cagggcaatt ctctgatcaa ggagaacatg aggatgaaag tggttatgga
     2401 agggtctgta aatgggcatc agttcaagtg tacgggggag ggcgaaggca atccctacat
     2461 ggggacccag accatgagga ttaaagtgat tgaagggggc cctctgcctt ttgcgttcga
     2521 tattctggct acgtcatttg gcggcggtag tcgcacgttc atcaaatacc ccaagggtat
     2581 acccgacttt ttcaaacaga gctttcctga aggttttacc tgggagcgag taacccggta
     2641 tgaagatgga ggggtagtga cggtgatgca ggataccagc cttgaagatg ggtgcctcgt
     2701 ctaccacgtg caagtgagag gcgtcaactt tcctagcaac ggcccagtga tgcagaagaa
     2761 gacaaaaggc tgggaaccaa atacagagat gatgtatccc gccgacgggg gcctcagggg
     2821 atatactcac atggctctga aagttgatgg cggtggcaaa ccaattccta accccctgct
     2881 gggccttgac tctacccagc aacaaggaaa gcctatccca aatcctctcc ttggtctgga
     2941 cagcaccggt ggccagcagg gaggcgggaa accaatccca aaccctctgt tgggtttgga
     3001 ttctacccag cagcagggta aacctatacc gaaccctctc ctggggctgg actccacagg
     3061 gggtggacat cttagctgct cctttgtgac tacctacaga tccaagaaga cggtagggaa
     3121 tattaagatg cctggcattc acgcggtcga tcataggttg gagaggctgg aagaaagcga
     3181 taatgaaatg tttgtggtac agagagaaca cgcagtggct aagtttgccg ggttgggagg
     3241 aggcggacag ggcggcaagc ctattcctaa tcctctgctc gggttggata gcacacagca
     3301 gcagggaaag cccattccaa accccctcct gggcctggat agcacccagc agcaaggcaa
     3361 acccattccc aaccctctct tgggtctgga ctcaacctac ggcgcgccag agggcagagg
     3421 aagtcttcta acatgcggtg acgtggagga gaatcccggc cctttaatta actctagact
     3481 ggacaagagc aaagtcataa actctgctct ggaattactc aatgaagtcg gtatcgaagg
     3541 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
     3601 cgtgaagaac aagcgggccc tgctcgatgc cctggcaatc gagatgctgg acaggcatca
     3661 tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
     3721 gtcattccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
     3781 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     3841 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     3901 cgtattggag gatcaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     3961 ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccatcagg gagccgaacc
     4021 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gaaaaacagc taaagtgcga
     4081 aagcggcgga ccggccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
     4141 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
     4201 catgctcccc gggtaagcta gcaattccgc tcgagataat caacctctgg attacaaaat
     4261 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     4321 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     4381 gtataaatcc tggttagttc ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     4441 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgtttattt gtgaaatttg
     4501 tgatgctatt gctttatttg taaccatcta gctttatttg tgaaatttgt gatgctattg
     4561 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     4621 ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcggccgc aggaacccct
     4681 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4741 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4801 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4861 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4921 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4981 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5041 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5101 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5161 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     5221 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     5281 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5341 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5401 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5461 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5521 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5581 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5641 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5701 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5761 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5821 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5881 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5941 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6001 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6061 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6121 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6181 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6241 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6301 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6361 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6421 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6481 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6541 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6601 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6661 gtgaa
//
