LOCUS       sequence-486965-        6904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-T7 in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6904)
  AUTHORS   E11 Bio
  TITLE     E11 Bio PRISM Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6904)
  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            "Unpublished"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-T7
FEATURES             Location/Qualifiers
     source          1..6904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      588..597
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      849..858
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    865..898
                     /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)."
     protein_bind    949..982
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1007..1595
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1060..1080)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1478..1489)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    1627..2103
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1826..1845)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2143..2283
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2143..2272
                     /label=AAV2 ITR
     rep_origin      2358..2813
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2445..2464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2655..2676
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2830..2849)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2949..2971
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3009..3027)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3095..3199
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3200..3268,3269..4060)
                     /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(3418..3437)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4231..4819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4720..4739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   4881..5010
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    5020..5141
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5057..5076)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5111..5130
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5165..5173,5174..5209,5210..5212,5213..5248,
                     5249..5251,5252..5287,5288..5293,5294..5911))
                     /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"
     protein_bind    6367..6385
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6404..6422
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6441..6459
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6478..6496
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6515..6533
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6552..6570
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6589..6607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6626..6644
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6666..6690
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(6789..6822)
                     /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)."
     protein_bind    complement(join(6873..6904,1..2))
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 atggcgcgcc tcacttgtcc aaggaattct tactttgatt atcgataagc ttgacctggg
       61 tgagacgtaa tgaatgctga acgcaccagt cctactgaag aattcttagc ccatctgttg
      121 accgccggtc atgcttgcca tactgccgcc cagctcgtcc atgcccatca catcggtaaa
      181 ggccttttgc cactccttga agttgagctc ggtcttggag tgcttgagct ccgtcttacg
      241 gaacacgtac atcggctggt tcttcagata gttagccgcc attggcttgg caaaggtgta
      301 ggtggtccgc gcagtgctcc ggtagcgctt gccatttcca gtggtgtaac tccacttaaa
      361 ggtactgatg atggttttgt cgttggggta agtcttcttc gacctgcacc agtccgcagc
      421 ggtcagcgag ttggtcatca caggaccgtc agcagggaaa ccagtcccct tcacctgggc
      481 ctctcctttg atgtggcttc cctcgtaggt gtagcggtag ttaacagtaa gggaggcacc
      541 atcttcaaac tgcattgtgc gatggacttg gtatccggag ccatctacca tggcggcctg
      601 gaaaggcgac atcccgtcag ggtagggcag gtactgatgg aagccatacc cgatatgagg
      661 gaccagaatc cagggggaga actggaggtc acccttggtg gacttcaggt ttaactcctc
      721 ataaccatca tttggattgc cggtgccctg acccaccatg tcaaagtcca caccgttgat
      781 ggagccaaag atgtgtaact catgtgtcgc tgggagagag gccatgttat cctcctcgcc
      841 cttgctcacc atggtggcgc tagcataact tcgtataaag tatcctatac gaagttattt
      901 gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat aacttcgtat
      961 aatgtatgct atacgaagtt atgaattcga tatcaagctt atcgataatc aacctctgga
     1021 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     1081 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     1141 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     1201 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     1261 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     1321 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     1381 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac
     1441 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     1501 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     1561 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag
     1621 atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
     1681 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag
     1741 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg
     1801 aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa
     1861 tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc
     1921 gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag
     1981 agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac
     2041 ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc
     2101 cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg
     2161 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     2221 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     2281 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     2341 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2401 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     2461 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2521 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     2581 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2641 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     2701 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     2761 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     2821 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     2881 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     2941 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3001 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3061 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3121 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3181 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3241 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3301 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3361 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3421 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3481 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3541 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3601 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3661 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3721 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3781 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3841 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3901 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3961 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4021 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4081 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4141 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4201 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4261 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4321 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4381 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4441 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4501 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4561 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4621 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4681 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4741 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4801 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4861 ctggcctttt gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc
     4921 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
     4981 gagtggccaa ctccatcact aggggttcct gcggccgcaa acttgtttat tgcagcttat
     5041 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5101 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg agatccttac
     5161 ttagttaccc ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat
     5221 caaggtcaaa gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg
     5281 cgtcggccgg cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt
     5341 ccaggccgaa aaggaaggca ggttcggctc cctgatggtc gaacagctca attgcttgtc
     5401 tcagaagtgg gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat
     5461 gctcctgatc ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca
     5521 gtgcgttctc cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt
     5581 cgtactgttt ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga
     5641 ggagagcaca gcggaatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt
     5701 ccagggggca gaagtgggta tgatgcctgt ccagcatctc gattgccagg gcatcgagca
     5761 gggcccgctt gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag
     5821 cgagtttcct tgtcgtcagg ccttcgatac cgacttcatt gagtaattcc agagcagagt
     5881 ttatgacttt gctcttgtcc agtctagaca tggtgaattc gatatcaagc ttatcgatac
     5941 cgtccgccgc agcgcagatg gtcgcgcccg tgccccccta tctcgcgcct cgcgtggtgc
     6001 ggtccggctg ggccggcggc ggcgcggacg cgaccaaggt ggccgggaag gggagtttgc
     6061 gggggaccgg cgagtgacgt cagcgcgcct tcagtgctga ggcggcggtg gcgcgcgccg
     6121 ccaggcgggg gcgaaggcac tgtccgcggt gctgaagctg gcagtgcgca cgcgcctcgc
     6181 cgcatcctgt ttcccctccc cctctctgat aggggatgcg caatttgggg aatgggggtt
     6241 gggtgcttgt ccagtgggtc ggggtcggtc gtcaggtagg cacccccacc ccgcctcatc
     6301 ctggtcctaa aacccacttg cactcatacg cagggccctc tgcagtatca agtgccacct
     6361 gacgtctccc tatcagtgat agagaagtcg acacgtctcg agctccctat cagtgataga
     6421 gaaggtacgt ctagaacgtc tccctatcag tgatagagaa gtcgacacgt ctcgagctcc
     6481 ctatcagtga tagagaaggt acgtctagaa cgtctcccta tcagtgatag agaagtcgac
     6541 acgtctcgag ctccctatca gtgatagaga aggtacgtct agaacgtctc cctatcagtg
     6601 atagagaagt cgacacgtct cgagctccct atcagtgata gagaagctac cccctatata
     6661 agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
     6721 ctccatagaa gacaccggga ccgatccagc ctggatccgg taccggatcc tctagagtcg
     6781 actccggaat aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat
     6841 tgtagctgct attagcaata tgaaacctct taataacttc gtatagcata cattatacga
     6901 agtt
//
