LOCUS       sequence-393304-        8368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral overexpression of PA-mCherry-KFERQ fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8368)
  AUTHORS   Ho PW, Leung CT, Liu H, Pang SY, Lam CS, Xian J, Li L, Kung MH, 
            Ramsden DB, Ho SL
  TITLE     Age-dependent accumulation of oligomeric SNCA/alpha-synuclein from 
            impaired degradation in mutant LRRK2 knockin mouse model of 
            Parkinson disease: role for therapeutic activation of 
            chaperone-mediated autophagy (CMA).
  JOURNAL   Autophagy. 2019 Apr 14:1-24. doi: 10.1080/15548627.2019.1603545.
  PUBMED    30983487
REFERENCE   2  (bases 1 to 8368)
  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: "Autophagy. 
            2019 Apr 14:1-24. doi: 10.1080/15548627.2019.1603545."
COMMENT     Sequence Label: pSIN-PAmCherry-KFERQ-NE
FEATURES             Location/Qualifiers
     source          1..8368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1187..1196
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1193..1900
                     /codon_start=1
                     /product="photoactivatable monomeric derivative of DsRed 
                     fluorescent protein (Subach et al., 2009)"
                     /label=PAmCherry1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFTWDILSPQFMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMKF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALK
                     GEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1338..1356)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    2186..2648
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2242..2259)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2469..2488
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2681..3280
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2681..2700)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3177..3197
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3483..3716
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(3867..3884)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3873..4097
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      4140..4149
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        4193..4297
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4298..4366,4367..5158)
                     /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(4516..4535)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5329..5917
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5818..5837
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             6076..6709
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6756..6881
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7374..7607
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7792..7836
                     /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    8123..8240
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattcgcca ccatggtgag
     1201 caagggcgag gaggataaca tggccatcat taaggagttc atgcgcttca aggtgcacat
     1261 ggaggggtcc gtgaacggcc acgtgttcga gatcgagggc gagggcgagg gccgccccta
     1321 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcacctg
     1381 ggacatcctg agccctcagt tcatgtacgg ctccaatgcc tacgtgaagc accccgccga
     1441 catccccgac tactttaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     1501 attcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     1561 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg tgatgcagaa
     1621 gaagaccatg ggctgggagg ccctctccga gcggatgtac cccgaggacg gcgccctgaa
     1681 gggcgaggtc aagcccagag tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     1741 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaaccgcaa
     1801 gctggacatc accagccaca acgaggacta caccatcgtg gagcagtacg agagagccga
     1861 gggccgccac tccaccggcg gcatggacga gctgtacaag gctagcaagg aatcccgggc
     1921 caagaaattc cagcggcagc atatggactc agacagttcc cccagcacca aagaaaaccc
     1981 tcgtagcaac caggaagaaa gctatgatga taacgaaagc taagtttaaa caatcactag
     2041 ttcgaaggat ccgcatgcat ctagggcggc caattccgcc cctctccccc ccccctaacg
     2101 ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca
     2161 ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga
     2221 gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga
     2281 aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca
     2341 ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag
     2401 atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa
     2461 gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac
     2521 cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga
     2581 ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac
     2641 gatgataagc ttgccacaac ccacaaggag acgaccttcc atgaccgagt acaagcccac
     2701 ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt
     2761 cgccgactac cccgccacgc gccacaccgt cgacccggac cgccacatcg agcgggtcac
     2821 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     2881 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     2941 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3001 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3061 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3121 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     3181 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     3241 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga taataggcgg ccgctcgaga
     3301 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc
     3361 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt
     3421 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
     3481 actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc
     3541 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     3601 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     3661 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag
     3721 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
     3781 tgagaggcct tgacattata atagatttag caggaattga actaggagtg gagcacacag
     3841 gcaaagttct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3901 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3961 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     4021 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     4081 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tgggggcgcg cccctcgagg
     4141 ccgccatggt catagctgtt tgacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4201 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcacga gacaataacc
     4261 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4321 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4381 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4441 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4501 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4561 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4621 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4681 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4741 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4801 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4861 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4921 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4981 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5041 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5101 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5161 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5221 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5281 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5341 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5401 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5461 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5521 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5581 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5641 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5701 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5761 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5821 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5881 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5941 acggttctgg ccttttgcac ggaccgccac tgccaattac ctgtggtttc atttactcta
     6001 aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt aaatatgtac
     6061 tacaaactta gtagttggaa gggctaattc actcccaaag aagacaagat atccttgatc
     6121 tgtggatcta ccacacacaa ggctacttcc ctgattagca gaactacaca ccagggccag
     6181 gggtcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata
     6241 aggtagaaga ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg
     6301 ggatggatga cccggagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc
     6361 atcacgtggc ccgagagctg catccggagt acttcaagaa ctgctgatat cgagcttgct
     6421 acaagggact ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt
     6481 ggcgagccct cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg
     6541 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     6601 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     6661 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
     6721 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
     6781 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
     6841 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     6901 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     6961 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     7021 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     7081 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     7141 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     7201 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     7261 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     7321 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     7381 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     7441 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     7501 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     7561 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     7621 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     7681 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     7741 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     7801 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     7861 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     7921 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     7981 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     8041 ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgccac
     8101 aaatggcagt attcatccac aattttaaaa gaaagggggg gattgggggg tacagtgcag
     8161 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     8221 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggatcg
     8281 ataagctttg caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac
     8341 cttctaggtc ttgaaaggag tgggaatt
//
