LOCUS       sequence-430538-        7837 bp    DNA     circular SYN 15-APR-2025
DEFINITION  All-in-one lentiviral construct for the dox repressible expression 
            of the A53T mutant of alpha-synuclein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7837)
  TITLE     Hartl Lab plasmids
REFERENCE   2  (bases 1 to 7837)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pTetOFF SNCA-A53T
FEATURES             Location/Qualifiers
     source          1..7837
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..118
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        172..486
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    180..198
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    216..234
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    251..269
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    287..305
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    323..341
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    358..376
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    394..412
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     459..483
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        934..1444
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1363..1381
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1454..1846
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1856..1909
                     /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(1910..2527,2528..2533,2534..2569,2570..2572,2573..2608,
                     2609..2611,2612..2647,2648..2656)
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(2696..2716)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3114..3125)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3330..3563
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3635..3756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3672..3691)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3726..3745
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3796..3931
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3931..4002
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4003..4071,4072..4863)
                     /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(4221..4240)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5034..5622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5523..5542
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5776..5793
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5910..5931
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5946..5951,5952..5969,5970..5976)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5984..6000
                     /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     5989..6011
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6008..6024
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6008..6024
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6043..6063
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6045..6063
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6091..6317
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6318..6498
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6545..6670
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7163..7396
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7581..7625
                     /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"
ORIGIN
        1 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
       61 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
      121 ggtttattac agggacagca gagatccact ttggccgcga atcgatatgt cgagtttact
      181 ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgatg
      241 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat
      301 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttatcc
      361 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
      421 cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag
      481 atcgcctgga gaattggcta gcaccatgga tgtattcatg aaaggacttt caaaggccaa
      541 ggagggagtt gtggctgctg ctgagaaaac caaacagggt gtggcagaag cagcaggaaa
      601 gacaaaagag ggtgttctct atgtaggctc caaaaccaag gagggagtgg tgcatggtgt
      661 gacaacagtg gctgagaaga ccaaagagca agtgacaaat gttggaggag cagtggtgac
      721 gggtgtgaca gcagtagccc agaagacagt ggagggagca gggagcattg cagcagccac
      781 tggctttgtc aaaaaggacc agttgggcaa gaatgaagaa ggagccccac aggaaggaat
      841 tctggaagat atgcctgtgg atcctgacaa tgaggcttat gaaatgcctt ctgaggaagg
      901 gtatcaagac tacgaacctg aagcctaagg tccggggttg gggttgcgcc ttttccaagg
      961 cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg
     1021 cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc
     1081 cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc
     1141 cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct
     1201 cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa
     1261 tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc
     1321 ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa
     1381 gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc
     1441 ccagcaattc accatggttc ctttgtctca agaagaatcc accctcattg aaagagcaac
     1501 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     1561 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     1621 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     1681 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     1741 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     1801 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggccccg gtgccgaagg
     1861 tagaggttct ctcctcactt gtggtgatgt tgaagaaaac cctggtccaa tgtctagact
     1921 ggacaagagc aaagtcataa actctgctct ggaattactc aatgaagtcg gtatcgaagg
     1981 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
     2041 cgtgaagaac aagcgggccc tgctcgatgc cctggcaatc gagatgctgg acaggcatca
     2101 tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
     2161 gtcattccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
     2221 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     2281 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     2341 cgtattggag gatcaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     2401 ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccatcagg gagccgaacc
     2461 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
     2521 aagcggcggg ccggccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
     2581 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
     2641 catgctcccc gggtaactaa gtaaggatcg atccaagata tcgtattctt aactatgttg
     2701 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     2761 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     2821 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     2881 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     2941 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3001 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     3061 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3121 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     3181 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctgtttcgc
     3241 ctcggcgtcc ggactagagg tacctttaag accaatgact tacaaggcag ctgtagatct
     3301 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     3361 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     3421 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     3481 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     3541 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     3601 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     3661 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3721 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     3781 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3841 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     3901 cagaagtagt gaggaggctt ttttggaggc ctttcaaata tgtatccgct catgagacaa
     3961 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4021 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4081 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4141 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4201 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4261 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4321 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4381 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4441 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4501 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4561 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4621 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4681 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4741 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4801 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4861 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4921 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4981 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5041 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5101 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5161 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5221 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5281 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5341 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5401 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5461 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5521 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5581 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5641 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5701 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5761 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     5821 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5881 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     5941 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     6001 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     6061 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
     6121 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
     6181 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
     6241 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
     6301 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     6361 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     6421 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     6481 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
     6541 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
     6601 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
     6661 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
     6721 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
     6781 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     6841 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     6901 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     6961 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct
     7021 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     7081 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     7141 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     7201 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     7261 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     7321 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     7381 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     7441 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     7501 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     7561 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     7621 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     7681 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     7741 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacaat
     7801 tctcgacctc gagacaaatg gcagtattca tccacaa
//
