LOCUS       sequence-486990-        6898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-E2 in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6898)
  AUTHORS   E11 Bio
  TITLE     E11 Bio PRISM Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6898)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-BD-hSynTet-TRE-DIO-mNeonGreen-E2
FEATURES             Location/Qualifiers
     source          1..6898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     438..457
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(676..780)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     848..866
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(904..926)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1026..1045
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(1062..1517)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1199..1220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1411..1430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   1592..1732
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1603..1732
                     /label=AAV2 ITR
     polyA_signal    1772..2248
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     2030..2049
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    2280..2868
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             2386..2397
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2795..2815
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(2893..2926)
                     /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(2977..3010)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      3017..3026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3278..3287
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3867..3900
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3951..3984
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(4083..4107)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4129..4147
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4166..4184
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4203..4221
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4240..4258
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4277..4295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4314..4332
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4351..4369
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4388..4406
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(4862..5479,5480..5485,5486..5521,5522..5524,5525..5560,
                     5561..5563,5564..5599,5600..5608)
                     /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"
     polyA_signal    5632..5753
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5643..5662)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5697..5716
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5763..5892
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5954..6542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6034..6053)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6713..6898,1..606,607..675))
                     /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"
ORIGIN
        1 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
       61 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
      121 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
      181 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
      241 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
      301 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
      361 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
      421 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
      481 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
      541 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
      601 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
      661 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
      721 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      781 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
      841 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
      901 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
      961 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     1021 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat
     1081 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aatagaccga
     1141 aatcggcaaa atcccttata aatcaaaaga atagcccgag atagagttga gtgttgttcc
     1201 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1261 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc
     1321 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     1381 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa
     1441 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     1501 gccgctacag ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat accgcacaga
     1561 tgcgtaagga gaaaataccg catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc
     1621 actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     1681 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
     1741 tcggtccgca cgtggttacc tacaaaatca gaaggacagg gaagggagca gtggttcacg
     1801 cctgtaatcc cagcaatttg ggaggccaag gtgggtagat cacctgagat taggagttgg
     1861 agaccagcct ggccaatatg gtgaaacccc gtctctacca aaaaaacaaa aattagctga
     1921 gcctggtcat gcatgcctgg aatcccaaca actcgggagg ctgaggcagg agaatcgctt
     1981 gaacccagga ggcggagatt gcagtgagcc aagattgtgc cactgcactc cagcttggtt
     2041 cccaatagac cccgcaggcc ctacaggttg tcttcccaac ttgccccttg ctccatacca
     2101 cccccctcca ccccataata ttatagaagg acacctagtc agacaaaatg atgcaactta
     2161 attttattag gacaaggctg gtgggcactg gagtggcaac ttccagggcc aggagaggca
     2221 ctggggaggg gtcacaggga tgccacccgt agatctctcg agcagcgctc ggtatcgatg
     2281 cggggaggcg gcccaaaggg agatccgact cgtctgaggg cgaaggcgaa gacgcggaag
     2341 aggccgcaga gccggcagca ggccgcggga aggaaggtcc gctggattga gggccgaagg
     2401 gacgtagcag aaggacgtcc cgcgcagaat ccaggtggca acataggcga gcagccaagg
     2461 aaaggacgat gatttccccg acaacaccac ggaattgtca gtgcccaaca gccgagcccc
     2521 tgtccagcag cgggcaaggc aggcggcgat gagttccgcc gtggcaatag ggagggggaa
     2581 agcgaaagtc ccggaaagga gctgacaggt ggtggcaatg ccccaaccag tgggggttgc
     2641 gtcagcaaac acagtgcaca ccacgccacg ttgcctgaca acgggccaca actcctcata
     2701 aagagacagc aaccaggatt tatacaagga ggagaaaatg aaagccatac gggaagcaat
     2761 agcatgatac aaaggcatta aagcagcgta tccacatagc gtaaaaggag caacatagtt
     2821 aagaatacca gtcaatcttt cacaaatttt gtaatccaga ggttgattat cgataagctt
     2881 gatatcgaat tcataacttc gtatagcata cattatacga agttattctt tgcaccattc
     2941 taaagaataa cagtgataat ttctgggtta aggcaaataa cttcgtatag gatactttat
     3001 acgaagttat gctagcgcca ccatggtgag caagggcgag gaggataaca tggcctctct
     3061 cccagcgaca catgagttac acatctttgg ctccatcaac ggtgtggact ttgacatggt
     3121 gggtcagggc accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa
     3181 gggtgacctc cagttctccc cctggattct ggtccctcat atcgggtatg gcttccatca
     3241 gtacctgccc taccctgacg ggatgtcgcc tttccaggcc gccatggtag atggctccgg
     3301 ataccaagtc catcgcacaa tgcagtttga agatggtgcc tcccttactg ttaactaccg
     3361 ctacacctac gagggaagcc acatcaaagg agaggcccag gtgaagggga ctggtttccc
     3421 tgctgacggt cctgtgatga ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa
     3481 gacttacccc aacgacaaaa ccatcatcag tacctttaag tggagttaca ccactggaaa
     3541 tggcaagcgc taccggagca ctgcgcggac cacctacacc tttgccaagc caatggcggc
     3601 taactatctg aagaaccagc cgatgtacgt gttccgtaag acggagctca agcactccaa
     3661 gaccgagctc aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg gcatggacga
     3721 gytgggcggc agcagcacca gcagcgactt cagagacaga taagaattct tcagtaggac
     3781 tggtaggctt tactacaatg cgtgggctcc caggtcaagc ttatcgataa tcaaagtaag
     3841 aattccttgg acaagtgagg cgcgccataa cttcgtataa tgtatgctat acgaagttat
     3901 taagaggttt catattgcta atagcagcta caatccagct accattctgc ataacttcgt
     3961 ataaagtatc ctatacgaag ttattccgga gtcgactcta gaggatccgg taccggatcc
     4021 aggctggatc ggtcccggtg tcttctatgg aggtcaaaac agcgtggatg gcgtctccag
     4081 gcgatctgac ggttcactaa acgagctctg cttatatagg gggtagcttc tctatcactg
     4141 atagggagct cgagacgtgt cgacttctct atcactgata gggagacgtt ctagacgtac
     4201 cttctctatc actgataggg agctcgagac gtgtcgactt ctctatcact gatagggaga
     4261 cgttctagac gtaccttctc tatcactgat agggagctcg agacgtgtcg acttctctat
     4321 cactgatagg gagacgttct agacgtacct tctctatcac tgatagggag ctcgagacgt
     4381 gtcgacttct ctatcactga tagggagacg tcaggtggca cttgatactg cagagggccc
     4441 tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc ggggtggggg tgcctacctg
     4501 acgaccgacc ccgacccact ggacaagcac ccaaccccca ttccccaaat tgcgcatccc
     4561 ctatcagaga gggggagggg aaacaggatg cggcgaggcg cgtgcgcact gccagcttca
     4621 gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc gccaccgccg cctcagcact
     4681 gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc cccttcccgg ccaccttggt
     4741 cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga gatagggggg
     4801 cacgggcgcg accatctgcg ctgcggcgga cggtatcgat aagcttgata tcgaattcac
     4861 catgtctaga ctggacaaga gcaaagtcat aaactctgct ctggaattac tcaatgaagt
     4921 cggtatcgaa ggcctgacga caaggaaact cgctcaaaag ctgggagttg agcagcctac
     4981 cctgtactgg cacgtgaaga acaagcgggc cctgctcgat gccctggcaa tcgagatgct
     5041 ggacaggcat catacccact tctgccccct ggaaggcgag tcatggcaag actttctgcg
     5101 gaacaacgcc aagtcattcc gctgtgctct cctctcacat cgcgacgggg ctaaagtgca
     5161 tctcggcacc cgcccaacag agaaacagta cgaaaccctg gaaaatcagc tcgcgttcct
     5221 gtgtcagcaa ggcttctccc tggagaacgc actgtacgct ctgtccgccg tgggccactt
     5281 tacactgggc tgcgtattgg aggatcagga gcatcaagta gcaaaagagg aaagagagac
     5341 acctaccacc gattctatgc ccccacttct gagacaagca attgagctgt tcgaccatca
     5401 gggagccgaa cctgccttcc ttttcggcct ggaactaatc atatgtggcc tggagaaaca
     5461 gctaaagtgc gaaagcggcg ggccggccga cgcccttgac gattttgact tagacatgct
     5521 cccagccgat gcccttgacg actttgacct tgatatgctg cctgctgacg ctcttgacga
     5581 ttttgacctt gacatgctcc ccgggtaact aagtaaggat ctcagacatg ataagataca
     5641 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     5701 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gtttgcggcc
     5761 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5821 ggccgggcga ccaaaggtcg cccgacgccc gggcggcctc agtgagcgag cgagcgcgca
     5881 gctgcctgca ggacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5941 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6001 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6061 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6121 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6181 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6241 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6301 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6361 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6421 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6481 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6541 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6601 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6661 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6721 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6781 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6841 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagcc
//
