LOCUS       sequence-486962-        6907 bp    DNA     circular SYN 23-SEP-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-Tag100 in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6907)
  AUTHORS   n/a
  TITLE     E11 Bio PRISM Collection Plasmids 2025
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6907)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 23, 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            "unpublished"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-Tag100
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(95..114)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      908..1496
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1397..1416
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1558..1687
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1697..1818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1734..1753)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1788..1807
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1842..1850,1851..1886,1887..1889,1890..1925,
                     1926..1928,1929..1964,1965..1970,1971..2588))
                     /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    3044..3062
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3081..3099
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3118..3136
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3155..3173
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3192..3210
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3229..3247
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3266..3284
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3303..3321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3343..3367
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3466..3499)
                     /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(3550..3583)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3690..3725)
                     /codon_start=1
                     /product="epitope tag derived from MAP kinase 2"
                     /label=Tag-100
                     /translation="EETARFQPGYRS"
     regulatory      4172..4181
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4433..4442
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4449..4482
                     /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    4533..4566
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4591..5179
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4644..4664)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5062..5073)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5211..5687
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5410..5429)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5727..5867
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5727..5856
                     /label=AAV2 ITR
     rep_origin      5942..6397
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6029..6048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6239..6260
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6414..6433)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6533..6555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6593..6611)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6679..6783
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6784..6852,6853..6907,1..737)
                     /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 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
       61 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
      121 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
      181 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
      241 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
      301 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
      361 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
      421 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
      481 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
      541 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
      601 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
      661 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
      721 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
      781 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
      841 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
      901 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
      961 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1021 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     1081 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1141 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1201 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1261 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1321 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1381 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1441 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1501 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct
     1561 gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt
     1621 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
     1681 ggttcctgcg gccgcaaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1801 actcatcaat gtatcttatc atgtctgaga tccttactta gttacccggg gagcatgtca
     1861 aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa ggtcaaagtc gtcaagggca
     1921 tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt cggccggccc gccgctttcg
     1981 cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag gaaggcaggt
     2041 tcggctccct gatggtcgaa cagctcaatt gcttgtctca gaagtggggg catagaatcg
     2101 gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgatcctc caatacgcag
     2161 cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag ggagaagcct
     2221 tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc tgttgggcgg
     2281 gtgccgagat gcactttagc cccgtcgcga tgtgagagga gagcacagcg gaatgacttg
     2341 gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagaa gtgggtatga
     2401 tgcctgtcca gcatctcgat tgccagggca tcgagcaggg cccgcttgtt cttcacgtgc
     2461 cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt cgtcaggcct
     2521 tcgataccga cttcattgag taattccaga gcagagttta tgactttgct cttgtccagt
     2581 ctagacatgg tgaattcgat atcaagctta tcgataccgt ccgccgcagc gcagatggtc
     2641 gcgcccgtgc ccccctatct cgcgcctcgc gtggtgcggt ccggctgggc cggcggcggc
     2701 gcggacgcga ccaaggtggc cgggaagggg agtttgcggg ggaccggcga gtgacgtcag
     2761 cgcgccttca gtgctgaggc ggcggtggcg cgcgccgcca ggcgggggcg aaggcactgt
     2821 ccgcggtgct gaagctggca gtgcgcacgc gcctcgccgc atcctgtttc ccctccccct
     2881 ctctgatagg ggatgcgcaa tttggggaat gggggttggg tgcttgtcca gtgggtcggg
     2941 gtcggtcgtc aggtaggcac ccccaccccg cctcatcctg gtcctaaaac ccacttgcac
     3001 tcatacgcag ggccctctgc agtatcaagt gccacctgac gtctccctat cagtgataga
     3061 gaagtcgaca cgtctcgagc tccctatcag tgatagagaa ggtacgtcta gaacgtctcc
     3121 ctatcagtga tagagaagtc gacacgtctc gagctcccta tcagtgatag agaaggtacg
     3181 tctagaacgt ctccctatca gtgatagaga agtcgacacg tctcgagctc cctatcagtg
     3241 atagagaagg tacgtctaga acgtctccct atcagtgata gagaagtcga cacgtctcga
     3301 gctccctatc agtgatagag aagctacccc ctatataagc agagctcgtt tagtgaaccg
     3361 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
     3421 atccagcctg gatccggtac cggatcctct agagtcgact ccggaataac ttcgtatagg
     3481 atactttata cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga
     3541 aacctcttaa taacttcgta tagcatacat tatacgaagt tatggcgcgc ctcacttgtc
     3601 caaggaattc ttactttgat tatcgataag cttgacctgg ctccactccg gcgtgctaag
     3661 gttacaccag tcctactgaa gaattcttag cttctgtagc caggctggaa tctggcggtc
     3721 tcctcgctgc cgcccagctc gtccatgccc atcacatcgg taaaggcctt ttgccactcc
     3781 ttgaagttga gctcggtctt ggagtgcttg agctccgtct tacggaacac gtacatcggc
     3841 tggttcttca gatagttagc cgccattggc ttggcaaagg tgtaggtggt ccgcgcagtg
     3901 ctccggtagc gcttgccatt tccagtggtg taactccact taaaggtact gatgatggtt
     3961 ttgtcgttgg ggtaagtctt cttcgacctg caccagtccg cagcggtcag cgagttggtc
     4021 atcacaggac cgtcagcagg gaaaccagtc cccttcacct gggcctctcc tttgatgtgg
     4081 cttccctcgt aggtgtagcg gtagttaaca gtaagggagg caccatcttc aaactgcatt
     4141 gtgcgatgga cttggtatcc ggagccatct accatggcgg cctggaaagg cgacatcccg
     4201 tcagggtagg gcaggtactg atggaagcca tacccgatat gagggaccag aatccagggg
     4261 gagaactgga ggtcaccctt ggtggacttc aggtttaact cctcataacc atcatttgga
     4321 ttgccggtgc cctgacccac catgtcaaag tccacaccgt tgatggagcc aaagatgtgt
     4381 aactcatgtg tcgctgggag agaggccatg ttatcctcct cgcccttgct caccatggtg
     4441 gcgctagcat aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat
     4501 tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga
     4561 agttatgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     4621 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     4681 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     4741 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     4801 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     4861 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     4921 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     4981 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     5041 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     5101 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     5161 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     5221 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     5281 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     5341 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     5401 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     5461 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     5521 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     5581 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     5641 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     5701 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     5761 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     5821 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     5881 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     5941 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     6001 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     6061 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     6121 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     6181 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     6241 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     6301 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     6361 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     6421 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     6481 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     6541 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     6601 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     6661 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     6721 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     6781 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     6841 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6901 gcacgag
//
