LOCUS       Exported                7993 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pAAV-tTS, complete sequence.
ACCESSION   JX006096
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7993)
  AUTHORS   Chen Y, Cao L, Luo C, Ditzel DA, Peter J, Sprengel R.
  TITLE     RANGE: Gene Transfer of Reversibly Controlled Polycistronic Genes
  JOURNAL   Mol Ther Nucleic Acids 2, E85 (2013)
  PUBMED    23571608
REFERENCE   2  (bases 1 to 7993)
  AUTHORS   Chen Y.
  TITLE     Direct Submission
  JOURNAL   Submitted (01-MAY-2012) Molecular Neurobiology, Max Planck Institute
            for Medical Research, Jahnstr. 29, Heidelberg, BW 69120, Germany
REFERENCE   3  (bases 1 to 7993)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7993
                     /organism="Cloning vector pAAV-tTS"
                     /mol_type="other DNA"
                     /db_xref="taxon:1286611"
     repeat_region   19..163
                     /rpt_family="AAV2 ITR"
     polyA_signal    248..296
                     /note="synthetic polyadenylation signal"
     regulatory      371..822
                     /regulatory_class="promoter"
                     /note="human synapsin"
     promoter        371..818
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             827..4117
                     /codon_start=1
                     /product="luciferase/tTS fusion"
                     /label=luciferase/tTS fusion
                     /protein_id="AGE09537.1"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLLEGRGSLLTCGDVEENPGPAPGSMSR
                     LDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVRNKQTLMNMLSEAILAK
                     HHTRSAPLPTESWQQFLQENALSFRKALLVHRDGARLHIGTSPTPPQFEQAEAQLRCLC
                     DAGFSVEEALFILQSISHFTLGAVLEEQATNQIENNHVIDAAPPLLQEAFNIQARTSAE
                     MAFHFGLKSLIFGFSAQLDEKKHTPIEDGNKPKKKRKLAVSVTFEDVAVLFTRDEWKKL
                     DLSQRSLYREVMLENYSNLASMAGFLFTKPKVISLLQQGEDPYLEGRGSLLTCGDVEEN
                     PGPAPGSMVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAKGGP
                     MPFAFDLVSHVFCYGHRPFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSASVSAHIS
                     LRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGG
                     NHKCQFKTTYKAAKKILKMPGSHYISHRLVRKTEGNITELVEDAVAHS"
     mat_peptide     827..2479
                     /product="firefly luciferase"
                     /EC_number="1.13.12.7"
     CDS             827..2476
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     mat_peptide     2480..2533
                     /product="2A peptide"
                     /note="self-cleaving"
     CDS             2480..2533
                     /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"
     mat_peptide     2546..3178
                     /product="TetR"
     misc_feature    3179..3196
                     /label=nuclear localization signal
                     /note="nuclear localization signal"
     mat_peptide     3197..3394
                     /product="kid-1"
     CDS             3203..3328
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the rat zinc finger protein Kid-1 
                     (Witzgall et al., 1994)"
                     /label=KRAB-A
                     /translation="VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASM"
     mat_peptide     3395..3448
                     /product="2A peptide"
     CDS             3395..3448
                     /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             3461..4117
                     /codon_start=1
                     /product="monomeric Kusabira-Orange fluorescent protein 
                     (Karasawa et al., 2004)"
                     /label=mKO
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAKGGPMPFAFDLVSHVFCYGHRPFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQFKTTYKAAKKILKMPGSHYISHRLVRKTEGNITELVEDAVAHS"
     mat_peptide     3461..4114
                     /product="Kusabira-orange fluorescent protein"
     misc_feature    4148..4744
                     /note="woodchuck posttranscriptional regulatory element 
                     (WRPE)"
     misc_feature    4150..4738
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(4621..4632)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     regulatory      4777..4987
                     /regulatory_class="polyA_signal_sequence"
                     /note="bovine growth hormone polyadenylation signal (BGH 
                     pA)"
     polyA_signal    4780..4987
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4996..5140
                     /rpt_family="AAV2 ITR"
     rep_origin      5239..5694
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        6026..6130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6131..6991
                     /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"
     rep_origin      7162..7750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"