LOCUS       sequence-358824-        6541 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cre-dependent expression of mCherry and a shRNA against luciferase 
            under the constitutive promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6541)
  AUTHORS   Wang LL, Serrano C, Zhong X, Ma S, Zou Y, Zhang CL
  TITLE     Revisiting astrocyte to neuron conversion with lineage tracing in 
            vivo.
  JOURNAL   Cell. 2021 Oct 14;184(21):5465-5481.e16. doi: 
            10.1016/j.cell.2021.09.005. Epub 2021 Sep 27.
  PUBMED    34582787
REFERENCE   2  (bases 1 to 6541)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2021.09.005"; journalName: "Cell"; date: 
            "2021-10-14- 14"; volume: "184"; issue: "21"; pages: "5465-5481.e16"
COMMENT     Sequence Label: AAV-CAG-LSL-mCherry-shLuci
FEATURES             Location/Qualifiers
     source          1..6541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     polyA_signal    292..413
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    456..1044
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             562..573
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     971..991
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     ncRNA           complement(1228..1242)
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     CDS             complement(1440..2150)
                     /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(1718..1737)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1987..2005
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2147..2156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2188..2221)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2250..2384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2504..2638
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2805..2838)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2862..2895)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2944..2963)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3025..3047)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     repeat_region   3899..4028
                     /label=AAV2 ITR
     rep_origin      complement(4265..4853)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4345..4364)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5024..5815,5816..5884))
                     /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     5647..5666
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5885..5989)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6015..6470)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6152..6173)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6364..6383
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagatcc ttatcgattt taccacattt gtagaggttt tacttgcttt aaaaaacctc
      241 ccacatctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
      301 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
      361 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
      421 tgctcgaagc ggccgcccgg gtcgacggta tcgatgcggg gaggcggccc aaagggagat
      481 ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg gcagcaggcc
      541 gcgggaagga aggtccgctg gattgagggc cgaagggacg tagcagaagg acgtcccgcg
      601 cagaatccag gtggcaacac aggcgagcag ccaaggaaag gacgatgatt tccccgacaa
      661 caccacggaa ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg caaggcaggc
      721 ggcgatgagt tccgccgtgg caatagggag ggggaaagcg aaagtcccgg aaaggagctg
      781 acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag tgcacaccac
      841 gccacgttgc ctgacaacgg gccacaactc ctcataaaga gacagcaacc aggatttata
      901 caaggaggag aaaatgaaag ccatacggga agcaatagca tgatacaaag gcattaaagc
      961 agcgtatcca catagcgtaa aaggagcaac atagttaaga ataccagtca atctttcaca
     1021 aattttgtaa tccagaggtt gattatcgat aagcttgatc cctcgacgga tccaaagtga
     1081 tttaatttat accattttaa ttcagctttg taaaaatgta tcaaagagat agcaaggtat
     1141 tcagttttag taaacaagat aattgctcga attctagccc cttgaagtcc gaggcagtag
     1201 gcataggaat tataatgctt atctatacat ctgtggcttc actatagata agcattataa
     1261 ttcctgcgct cactgtcaac agcaatatac cttctcgagc cttctgttgg gttaagaagt
     1321 cgaagtaatc ccagcaagtg tttccaagat gtgcaggcaa cgattctgta aagtactgaa
     1381 gcctcattca acatagtata tgtgctgccg aagcgagcac ttaacaaggc ttgcggccgc
     1441 tacttgtaca gctcgtccat gccgccggtg gagtggcggc cctcggcgcg ttcgtactgt
     1501 tccacgatgg tgtagtcctc gttgtgggag gtgatgtcca acttgatgtt gacgttgtag
     1561 gcgccgggca gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc agcgtcgtag
     1621 tggccgccgt ccttcagctt cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg
     1681 gggtacatcc gctcggagga ggcctcccag cccatggtct tcttctgcat tacggggccg
     1741 tcggagggga agttggtgcc gcgcagcttc accttgtaga tgaactcgcc gtcctgcagg
     1801 gaggagtcct gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac
     1861 ttgaagccct cggggaagga cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg
     1921 taggccttgg agccgtacat gaactgaggg gacaggatgt cccaggcgaa gggcaggggg
     1981 ccacccttgg tcaccttcag cttggcggtc tgggtgccct cgtaggggcg gccctcgccc
     2041 tcgccctcga tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc
     2101 atgaactcct tgatgatggc catgttatcc tcctcgccct tgctcaccat ggtggcaccg
     2161 gtcttgggct gcaggtcgag ggacctaata acttcgtata gcatacatta tacgaagtta
     2221 tattaagggt tccggatcag cttgatgggg atccagacat gataagatac attgatgagt
     2281 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     2341 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     2401 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     2461 tctacaaatg tggtatggct gattatgatc ctctagagtc gcagatccag acatgataag
     2521 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     2581 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     2641 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     2701 aagcaagtaa aacctctaca aatgtggtat ggctgattat gatcctctag agtcgcagat
     2761 cctctagagt cgcagatctg caagctttca tttattcatc gcgaataact tcgtatagca
     2821 tacattatac gaagttatat ttaaatgatt taaatgtcga cgaagttcct atactttcta
     2881 gagaatagga acttcttagg gcccgcggta tcaaatcaat tctttgccaa aatgatgaga
     2941 cagcacaata accagcacgt tgcccaggag ctgtaggaaa aagaagaagg catgaacatg
     3001 gttagcagag gcgctagagc cgccggtcac acgccagaag ccgaaccccg ccctgccccg
     3061 tcccccccga aggcagccgt ccccccgcgg acagccccga gccgctcccc ccgcacaaag
     3121 ggccctcccg gagcccctca aggctttcac gcagccacag aaaagaaaca agccgtcatt
     3181 aaaccaagcg ctaattacag cccggaggag aagggccgtc ccgcccgctc acctgtggga
     3241 gtaacgcggt cagtcagagc cggggcgggc ggcgcgaggc ggcggcggag cggggcacgg
     3301 ggcgaaggca gcgcgcagcg actcccgccc gccgcgcgct tcgcttttta tagggccgcc
     3361 gccgccgccg cctcgccata aaaggaaact ttcggagcgc gccgctctga ttggctgccg
     3421 ccgcacctct ccgcctcgcc ccgccccgcc cctcgccccg ccccgccccc cccccccccc
     3481 ccgcccccat cgctgcacaa aataattaaa aaataaataa atacaaaatt gggggtgggg
     3541 agggggggga gatggggaga gtgaagcaga acgtggggct cacctcgacc atggtaatag
     3601 cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg
     3661 cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat
     3721 gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc
     3781 aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac gtcaatggta
     3841 ctcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag
     3901 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     3961 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4021 gcgcgcagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4081 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4141 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4201 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4261 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4321 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4381 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4441 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4501 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4561 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4621 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4681 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4741 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4801 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4861 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4921 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4981 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5041 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5101 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5161 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5221 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5281 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5341 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5401 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5461 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5521 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5581 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5641 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5701 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5761 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5821 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5881 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5941 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6001 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     6061 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     6121 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     6181 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     6241 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     6301 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     6361 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     6421 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     6481 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     6541 g
//
