LOCUS       pAAV-TRE-fDIO-me        7176 bp DNA     circular SYN 01-APR-2025
DEFINITION  Amplifier AAV vector of Dual-AAV sparse labeling system encoding 
            membrane-tethered seTurboID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7176)
  AUTHORS   Shilin Zhong
  TITLE     Direct Submission
  JOURNAL   Exported Apr 1, 2025 from SnapGene 7.1.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7176
                     /mol_type="other DNA"
                     /note="color: #ffffff"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     promoter        159..473
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven
                     tet operator sequences followed by the minimal CMV 
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     protein_bind    167..185
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
     protein_bind    203..221
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
     protein_bind    238..256
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
     protein_bind    274..292
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
     protein_bind    310..328
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
     protein_bind    345..363
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
     protein_bind    381..399
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
     protein_bind    504..551
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
                     /note="color: #31849b; direction: RIGHT"
     protein_bind    560..607
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="color: #31849b; direction: RIGHT"
     CDS             complement(join(616..624,625..660,661..663,664..699,
                     700..702,703..738,739..744,745..1362))
                     /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."
                     /note="This feature has 8 segments:
                        1:  616 ..  624 / #cc99b2
                        2:  625 ..  660 / #993366 / VP16 minimal transcriptio...
                        3:  661 ..  663 / #------
                        4:  664 ..  699 / #993366 / VP16 minimal transcriptio...
                        5:  700 ..  702 / #------
                        6:  703 ..  738 / #993366 / VP16 minimal transcriptio...
                        7:  739 ..  744 / #------
                        8:  745 .. 1362 / #993366 / TetR"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    1363..1946
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
                     /note="color: #a6acb3"
     CDS             complement(1951..3183)
                     /codon_start=1
                     /label=mem-seTurboID
                     /note="color: #993366"
                     /translation="MLCCMRRTKQVEKNDEDQKIDYKDDDDKGSMASKDNTVPLKLIAL
                     LANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQI
                     LGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANL
                     YLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVEL
                     AGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAAL
                     ELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPW
                     MGGEISLRSAEKGGGGSYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWT
                     YDDATKTFTVTE"
     CDS             complement(3100..3123)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /label=FLAG
                     /note="color: #cc99b2"
                     /translation="DYKDDDDK"
     protein_bind    complement(3196..3243)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
                     /note="color: #31849b; direction: LEFT"
     protein_bind    complement(3245..3292)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="color: #008080; direction: LEFT"
     misc_feature    3316..3904
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
                     /note="color: #ffffff"
     CDS             complement(3787..3798)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2
                     Cleavage site after base 3786"
                     /translation="IEGR"
     polyA_signal    3923..4399
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     repeat_region   4439..4579
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     rep_origin      4654..5109
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        5391..5495
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             join(5496..5564,5565..6356)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 5496 .. 5564 / #ccffcc / signal sequence
                        2: 5565 .. 6356 / #ccffcc"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6527..7115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtctcga gtttactccc tatcagtgat
      181 agagaacgta tgtcgagttt actccctatc agtgatagag aacgatgtcg agtttactcc
      241 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
      301 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttatcccta tcagtgatag
      361 agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga ggtaggcgtg
      421 tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagaa
      481 ttctgtcgtg aggtaccgga tccgaagttc ctattccgaa gttcctattc tctagaaagt
      541 ataggaactt cttaattaag aagttcctat tccgaagttc ctattcttca aatagtatag
      601 gaacttcggc gcgccttacc cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc
      661 aggcagcata tcaaggtcaa agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa
      721 atcgtcaagg gcgtcggccg gcccgccgct ttcgcacttt agctgtttct ccaggccaca
      781 tatgattagt tccaggccga aaaggaaggc aggttcggct ccctgatggt cgaacagctc
      841 aattgcttgt ctcagaagtg ggggcataga atcggtggta ggtgtctctc tttcctcttt
      901 tgctacttga tgctcctgat cctccaatac gcagcccagt gtaaagtggc ccacggcgga
      961 cagagcgtac agtgcgttct ccagggagaa gccttgctga cacaggaacg cgagctgatt
     1021 ttccagggtt tcgtactgtt tctctgttgg gcgggtgccg agatgcactt tagccccgtc
     1081 gcgatgtgag aggagagcac agcggaatga cttggcgttg ttccgcagaa agtcttgcca
     1141 tgactcgcct tccagggggc agaagtgggt atgatgcctg tccagcatct cgattgccag
     1201 ggcatcgagc agggcccgct tgttcttcac gtgccagtac agggtaggct gctcaactcc
     1261 cagcttttga gcgagtttcc ttgtcgtcag gccttcgata ccgacttcat tgagtaattc
     1321 cagagcagag tttatgactt tgctcttgtc cagtctagac attgtggcca tattatcatc
     1381 gtgtttttca aaggaaaacc acgtccccgt ggttcggggg gcctagacgt ttttttaacc
     1441 tcgactaaac acatgtaaag catgtgtacc gaggccccag atcagatccc atacaatggg
     1501 gtaccttctg ggcatccttc agccccttgt tgaatacgct tgaggagagc catttgactc
     1561 tttccacaac tatccaactc acaacgtggc actggggttg tgccgccttt gcaggtgtat
     1621 cttatacacg tggcttttgg ccgcagaggc acctgtcgcc aggtgggggg ttccgctgcc
     1681 tgcaaagggt cgctacagac gttgtttgtc ttcaagaagc ttccagagga actgcttcct
     1741 tcacgacatt caacagacct tgcattcctt tggcgagagg ggaaagaccc ctaggaatgc
     1801 tcgtcaagaa gacagggcca ggtttccggg ccctcacatt gccaaaagac ggcaatatgg
     1861 tggaaaataa catatagaca aacgcacacc ggccttattc caagcggctt cggccagtaa
     1921 cgttaggggg gggggaggga gaggggctta ttcggttacc gtgaaggttt tggtagcgtc
     1981 gtcgtaggtc cattcaccgt caacaccgtt gtcgttagcg tactgtttga aaactttttc
     2041 cgcggtagca gcgtcaacag cttcggtggt ggtttcacct ttcagggttt taccgttcag
     2101 gataagcttg taagatccgc caccgccctt ttcggcagac cgcagactga tttctccgcc
     2161 catccagggt ttgataactc cgtcctgttc cagcagcagg gctccctgtt tatcaatccc
     2221 ccggctaatc ccgaaaatct ctttgtcccc aatgatcagc ttcacgggtc tattgatgaa
     2281 gttatccagc ttctcccacc gtggcagata tggagccagg ccttcctgct cgaacagttc
     2341 cagggctgcc cgcagctctc ggatcagcgt agcggccaga gtattcctgt ccaggttaat
     2401 ccctgcttcc tgcagtgtga tccagccctg attgaccacg ctttcctcca cgcgcctcat
     2461 agccacgtta atcccggcgc caatgacgat ctgtgcagca tcgcctgtta ttccggccag
     2521 ctccaccagg atgcctgcca gctttctatc ctgcagatac aggtcattgg gccatttgac
     2581 tcgcaccttg tctgctccca gctttcgcag cgcttctgcc atgacaattc cgatgaccgg
     2641 gcccaggccg attgctgctg gtccccgctt caggcgccaa aacatactca ggtacaggtt
     2701 agctccaaaa ggagagaacc atttcctccc tctgcttcct ctccctgcct gctgatattc
     2761 tgcaatgcaa gcatccccac tcttcagctc gccgattcga tccagcaggt actgattggt
     2821 ggagtcgacc acaggcagga ctgccacgct cccgccgtcc agctgtccca gaatctgttt
     2881 agcgttcagc agcgggatag gctcgggcag agagtagccc tttccgggca cggtgaacac
     2941 gtccacgccc cagtcgcgca gagtctgaat gtgcttgttg atagcggccc tggacatgcc
     3001 cagggtttct cccagctgtt cgccactatg gaactcgcca ttagccagga gagcgatcag
     3061 cttcagaggc acagtattgt ctttgctagc catggatccc ttatcgtcgt catccttgta
     3121 atcgatcttt tggtcctcat cattcttttc aacctgtttg gttcttctca tacagcacag
     3181 catggtggcg ctagcgaagt tcctatactt tctagagaat aggaacttcg gaataggaac
     3241 ttcggaagtt cctatactat ttgaagaata ggaacttcgg aataggaact tcaattcgat
     3301 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3361 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3421 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3481 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3541 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3601 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3661 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3721 tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3781 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3841 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     3901 ccgcatcgat accgagatct acgggtggca tccctgtgac ccctccccag tgcctctcct
     3961 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     4021 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     4081 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     4141 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     4201 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     4261 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     4321 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     4381 tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag
     4441 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4501 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4561 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4621 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     4681 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     4741 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4801 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4861 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4921 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4981 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     5041 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     5101 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     5161 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5221 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5281 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5341 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5401 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5461 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5521 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5581 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5641 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5701 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5761 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5821 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5881 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5941 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6001 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6061 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6121 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6181 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6241 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6301 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6361 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6421 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6481 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6541 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6601 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6661 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6721 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6781 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6841 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6901 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6961 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7021 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7081 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7141 ggttcctggc cttttgctgg ccttttgctc acatgt
//
