LOCUS       sequence-486974-        6898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-HA 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  JOURNAL   Unpublished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            "Unpublished"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-HA
FEATURES             Location/Qualifiers
     source          1..6898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..28
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(247..351)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     419..437
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(475..497)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     597..616
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(633..1088)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(770..791)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     982..1001
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   1163..1303
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1174..1303
                     /label=AAV2 ITR
     polyA_signal    1343..1819
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     1601..1620
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    1851..2439
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1957..1968
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2366..2386
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(2464..2497)
                     /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(2548..2581)
                     /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      2588..2597
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2849..2858
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3305..3331
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    3438..3471
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3522..3555
                     /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(3654..3678)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3700..3718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3737..3755
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3774..3792
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3811..3829
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3848..3866
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3885..3903
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3922..3940
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3959..3977
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(4433..5050,5051..5056,5057..5092,5093..5095,5096..5131,
                     5132..5134,5135..5170,5171..5179)
                     /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    5203..5324
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5214..5233)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5268..5287
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5334..5463
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5525..6113)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5605..5624)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6284..6898,1..177,178..246))
                     /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 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
       61 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
      121 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
      181 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
      241 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
      301 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
      361 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
      421 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
      481 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
      541 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
      601 atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata ttttgttaaa
      661 attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg aaatcggcaa
      721 aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc cagtttggaa
      781 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
      841 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
      901 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
      961 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta aggcgctggc
     1021 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     1081 gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag atgcgtaagg
     1141 agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct cactgaggcc
     1201 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
     1261 gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg ctcggtccgc
     1321 acgtggttac ctacaaaatc agaaggacag ggaagggagc agtggttcac gcctgtaatc
     1381 ccagcaattt gggaggccaa ggtgggtaga tcacctgaga ttaggagttg gagaccagcc
     1441 tggccaatat ggtgaaaccc cgtctctacc aaaaaaacaa aaattagctg agcctggtca
     1501 tgcatgcctg gaatcccaac aactcgggag gctgaggcag gagaatcgct tgaacccagg
     1561 aggcggagat tgcagtgagc caagattgtg ccactgcact ccagcttggt tcccaataga
     1621 ccccgcaggc cctacaggtt gtcttcccaa cttgcccctt gctccatacc acccccctcc
     1681 accccataat attatagaag gacacctagt cagacaaaat gatgcaactt aattttatta
     1741 ggacaaggct ggtgggcact ggagtggcaa cttccagggc caggagaggc actggggagg
     1801 ggtcacaggg atgccacccg tagatctctc gagcagcgct cggtatcgat gcggggaggc
     1861 ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag
     1921 agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag ggacgtagca
     1981 gaaggacgtc ccgcgcagaa tccaggtggc aacataggcg agcagccaag gaaaggacga
     2041 tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc ctgtccagca
     2101 gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga aagcgaaagt
     2161 cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg cgtcagcaaa
     2221 cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat aaagagacag
     2281 caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa tagcatgata
     2341 caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt taagaatacc
     2401 agtcaatctt tcacaaattt tgtaatccag aggttgatta tcgataagct tgatatcgaa
     2461 ttcataactt cgtatagcat acattatacg aagttattct ttgcaccatt ctaaagaata
     2521 acagtgataa tttctgggtt aaggcaaata acttcgtata ggatacttta tacgaagtta
     2581 tgctagcgcc accatggtga gcaagggcga ggaggataac atggcctctc tcccagcgac
     2641 acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg tgggtcaggg
     2701 caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca agggtgacct
     2761 ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc agtacctgcc
     2821 ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg gataccaagt
     2881 ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc gctacaccta
     2941 cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc ctgctgacgg
     3001 tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga agacttaccc
     3061 caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa atggcaagcg
     3121 ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg ctaactatct
     3181 gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca agaccgagct
     3241 caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg agctgggcgg
     3301 cagctaccct tacgacgtgc ctgactacgc ctaagaattc ttcagtagga ctggtttgac
     3361 atgtacgcca tttgggtcgc ccaggtcaag cttatcgata atcaaagtaa gaattccttg
     3421 gacaagtgag gcgcgccata acttcgtata atgtatgcta tacgaagtta ttaagaggtt
     3481 tcatattgct aatagcagct acaatccagc taccattctg cataacttcg tataaagtat
     3541 cctatacgaa gttattccgg agtcgactct agaggatccg gtaccggatc caggctggat
     3601 cggtcccggt gtcttctatg gaggtcaaaa cagcgtggat ggcgtctcca ggcgatctga
     3661 cggttcacta aacgagctct gcttatatag ggggtagctt ctctatcact gatagggagc
     3721 tcgagacgtg tcgacttctc tatcactgat agggagacgt tctagacgta ccttctctat
     3781 cactgatagg gagctcgaga cgtgtcgact tctctatcac tgatagggag acgttctaga
     3841 cgtaccttct ctatcactga tagggagctc gagacgtgtc gacttctcta tcactgatag
     3901 ggagacgttc tagacgtacc ttctctatca ctgataggga gctcgagacg tgtcgacttc
     3961 tctatcactg atagggagac gtcaggtggc acttgatact gcagagggcc ctgcgtatga
     4021 gtgcaagtgg gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac
     4081 cccgacccac tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag
     4141 agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg
     4201 acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg
     4261 ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc
     4321 gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc
     4381 gaccatctgc gctgcggcgg acggtatcga taagcttgat atcgaattca ccatgtctag
     4441 actggacaag agcaaagtca taaactctgc tctggaatta ctcaatgaag tcggtatcga
     4501 aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt gagcagccta ccctgtactg
     4561 gcacgtgaag aacaagcggg ccctgctcga tgccctggca atcgagatgc tggacaggca
     4621 tcatacccac ttctgccccc tggaaggcga gtcatggcaa gactttctgc ggaacaacgc
     4681 caagtcattc cgctgtgctc tcctctcaca tcgcgacggg gctaaagtgc atctcggcac
     4741 ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca
     4801 aggcttctcc ctggagaacg cactgtacgc tctgtccgcc gtgggccact ttacactggg
     4861 ctgcgtattg gaggatcagg agcatcaagt agcaaaagag gaaagagaga cacctaccac
     4921 cgattctatg cccccacttc tgagacaagc aattgagctg ttcgaccatc agggagccga
     4981 acctgccttc cttttcggcc tggaactaat catatgtggc ctggagaaac agctaaagtg
     5041 cgaaagcggc gggccggccg acgcccttga cgattttgac ttagacatgc tcccagccga
     5101 tgcccttgac gactttgacc ttgatatgct gcctgctgac gctcttgacg attttgacct
     5161 tgacatgctc cccgggtaac taagtaagga tctcagacat gataagatac attgatgagt
     5221 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     5281 ctattgcttt atttgtaacc attataagct gcaataaaca agtttgcggc cgcaggaacc
     5341 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     5401 accaaaggtc gcccgacgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     5461 aggacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5521 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5581 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5641 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5701 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5761 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5821 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5881 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5941 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6001 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6061 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6121 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6181 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6241 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6301 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6361 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6421 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6481 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6541 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6601 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6661 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6721 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6781 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6841 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtc
//
