LOCUS       sequence-486992-        6889 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-AU5 in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6889)
  AUTHORS   E11 Bio
  TITLE     E11 Bio PRISM Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6889)
  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            "Unpublished"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-AU5
FEATURES             Location/Qualifiers
     source          1..6889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(97..116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      910..1498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1399..1418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1560..1689
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1699..1820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1736..1755)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1790..1809
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1844..1852,1853..1888,1889..1891,1892..1927,
                     1928..1930,1931..1966,1967..1972,1973..2590))
                     /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    3046..3064
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3083..3101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3120..3138
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3157..3175
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3194..3212
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3231..3249
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3268..3286
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3305..3323
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3345..3369
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3468..3501)
                     /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(3552..3585)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4156..4165
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4417..4426
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4433..4466
                     /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    4517..4550
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4575..5163
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4628..4648)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5046..5057)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5195..5671
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5394..5413)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5711..5851
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5711..5840
                     /label=AAV2 ITR
     rep_origin      5926..6381
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6013..6032)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6223..6244
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6398..6417)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6517..6539
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6577..6595)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6663..6767
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6768..6836,6837..6889,1..739)
                     /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 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
       61 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      121 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      421 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
      781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
      841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
      901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
      961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1021 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc
     1561 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg
     1621 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
     1681 ggggttcctg cggccgcaaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     1741 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     1801 aaactcatca atgtatctta tcatgtctga gatccttact tagttacccg gggagcatgt
     1861 caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc aaggtcaaag tcgtcaaggg
     1921 catcggctgg gagcatgtct aagtcaaaat cgtcaagggc gtcggccggc ccgccgcttt
     1981 cgcactttag ctgtttctcc aggccacata tgattagttc caggccgaaa aggaaggcag
     2041 gttcggctcc ctgatggtcg aacagctcaa ttgcttgtct cagaagtggg ggcatagaat
     2101 cggtggtagg tgtctctctt tcctcttttg ctacttgatg ctcctgatcc tccaatacgc
     2161 agcccagtgt aaagtggccc acggcggaca gagcgtacag tgcgttctcc agggagaagc
     2221 cttgctgaca caggaacgcg agctgatttt ccagggtttc gtactgtttc tctgttgggc
     2281 gggtgccgag atgcacttta gccccgtcgc gatgtgagag gagagcacag cggaatgact
     2341 tggcgttgtt ccgcagaaag tcttgccatg actcgccttc cagggggcag aagtgggtat
     2401 gatgcctgtc cagcatctcg attgccaggg catcgagcag ggcccgcttg ttcttcacgt
     2461 gccagtacag ggtaggctgc tcaactccca gcttttgagc gagtttcctt gtcgtcaggc
     2521 cttcgatacc gacttcattg agtaattcca gagcagagtt tatgactttg ctcttgtcca
     2581 gtctagacat ggtgaattcg atatcaagct tatcgatacc gtccgccgca gcgcagatgg
     2641 tcgcgcccgt gcccccctat ctcgcgcctc gcgtggtgcg gtccggctgg gccggcggcg
     2701 gcgcggacgc gaccaaggtg gccgggaagg ggagtttgcg ggggaccggc gagtgacgtc
     2761 agcgcgcctt cagtgctgag gcggcggtgg cgcgcgccgc caggcggggg cgaaggcact
     2821 gtccgcggtg ctgaagctgg cagtgcgcac gcgcctcgcc gcatcctgtt tcccctcccc
     2881 ctctctgata ggggatgcgc aatttgggga atgggggttg ggtgcttgtc cagtgggtcg
     2941 gggtcggtcg tcaggtaggc acccccaccc cgcctcatcc tggtcctaaa acccacttgc
     3001 actcatacgc agggccctct gcagtatcaa gtgccacctg acgtctccct atcagtgata
     3061 gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc tagaacgtct
     3121 ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat agagaaggta
     3181 cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc tccctatcag
     3241 tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc gacacgtctc
     3301 gagctcccta tcagtgatag agaagctacc ccctatataa gcagagctcg tttagtgaac
     3361 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
     3421 cgatccagcc tggatccggt accggatcct ctagagtcga ctccggaata acttcgtata
     3481 ggatacttta tacgaagtta tgcagaatgg tagctggatt gtagctgcta ttagcaatat
     3541 gaaacctctt aataacttcg tatagcatac attatacgaa gttatggcgc gcctcacttg
     3601 tccaaggaat tcttactttg attatcgata agcttgacct ggttgtaagc tgaaccgcag
     3661 tcctcccacc agtcctactg aagaattctt acttcaggta gaagtcggtg ctgccgccca
     3721 actcgtccat gcccatcaca tcggtaaagg ccttttgcca ctccttgaag ttgagctcgg
     3781 tcttggagtg cttgagctcc gtcttacgga acacgtacat cggctggttc ttcagatagt
     3841 tagccgccat tggcttggca aaggtgtagg tggtccgcgc agtgctccgg tagcgcttgc
     3901 catttccagt ggtgtaactc cacttaaagg tactgatgat ggttttgtcg ttggggtaag
     3961 tcttcttcga cctgcaccag tccgcagcgg tcagcgagtt ggtcatcaca ggaccgtcag
     4021 cagggaaacc agtccccttc acctgggcct ctcctttgat gtggcttccc tcgtaggtgt
     4081 agcggtagtt aacagtaagg gaggcaccat cttcaaactg cattgtgcga tggacttggt
     4141 atccggagcc atctaccatg gcggcctgga aaggcgacat cccgtcaggg tagggcaggt
     4201 actgatggaa gccatacccg atatgaggga ccagaatcca gggggagaac tggaggtcac
     4261 ccttggtgga cttcaggttt aactcctcat aaccatcatt tggattgccg gtgccctgac
     4321 ccaccatgtc aaagtccaca ccgttgatgg agccaaagat gtgtaactca tgtgtcgctg
     4381 ggagagaggc catgttatcc tcctcgccct tgctcaccat ggtggcgcta gcataacttc
     4441 gtataaagta tcctatacga agttatttgc cttaacccag aaattatcac tgttattctt
     4501 tagaatggtg caaagaataa cttcgtataa tgtatgctat acgaagttat gaattcgata
     4561 tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     4621 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     4681 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     4741 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     4801 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     4861 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     4921 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     4981 ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     5041 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     5101 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     5161 cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc
     5221 agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa
     5281 attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg
     5341 ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg
     5401 ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt
     5461 tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag
     5521 gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt
     5581 ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca
     5641 ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc
     5701 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     5761 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     5821 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     5881 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     5941 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     6001 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     6061 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6121 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     6181 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6241 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     6301 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6361 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     6421 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     6481 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6541 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     6601 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     6661 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6721 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6781 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6841 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacg
//
