LOCUS       sequence-486960-        6904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-VSV 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  JOURNAL   Unpublished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            "Unpublished"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-VSV
FEATURES             Location/Qualifiers
     source          1..6904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(115..134)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      928..1516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1417..1436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1578..1707
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1717..1838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1754..1773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1808..1827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1862..1870,1871..1906,1907..1909,1910..1945,
                     1946..1948,1949..1984,1985..1990,1991..2608))
                     /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    3064..3082
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3101..3119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3138..3156
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3175..3193
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3212..3230
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3249..3267
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3286..3304
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3323..3341
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3363..3387
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3486..3519)
                     /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(3570..3603)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3710..3742)
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     regulatory      4189..4198
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4450..4459
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4466..4499
                     /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    4550..4583
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4608..5196
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4661..4681)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5079..5090)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5228..5704
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5427..5446)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5744..5884
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5744..5873
                     /label=AAV2 ITR
     rep_origin      5959..6414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6046..6065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6256..6277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6431..6450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6550..6572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6610..6628)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6696..6800
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6801..6869,6870..6904,1..757)
                     /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 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
       61 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      121 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      181 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      241 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
      301 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
      361 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
      421 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
      481 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
      541 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
      601 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      661 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      721 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
      781 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
      841 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
      901 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
      961 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1021 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1081 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1141 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1201 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1261 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1321 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1381 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1441 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1501 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1561 gccttttgct cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
     1621 ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag
     1681 tggccaactc catcactagg ggttcctgcg gccgcaaact tgtttattgc agcttataat
     1741 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1801 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgaga tccttactta
     1861 gttacccggg gagcatgtca aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa
     1921 ggtcaaagtc gtcaagggca tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt
     1981 cggccggccc gccgctttcg cactttagct gtttctccag gccacatatg attagttcca
     2041 ggccgaaaag gaaggcaggt tcggctccct gatggtcgaa cagctcaatt gcttgtctca
     2101 gaagtggggg catagaatcg gtggtaggtg tctctctttc ctcttttgct acttgatgct
     2161 cctgatcctc caatacgcag cccagtgtaa agtggcccac ggcggacaga gcgtacagtg
     2221 cgttctccag ggagaagcct tgctgacaca ggaacgcgag ctgattttcc agggtttcgt
     2281 actgtttctc tgttgggcgg gtgccgagat gcactttagc cccgtcgcga tgtgagagga
     2341 gagcacagcg gaatgacttg gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca
     2401 gggggcagaa gtgggtatga tgcctgtcca gcatctcgat tgccagggca tcgagcaggg
     2461 cccgcttgtt cttcacgtgc cagtacaggg taggctgctc aactcccagc ttttgagcga
     2521 gtttccttgt cgtcaggcct tcgataccga cttcattgag taattccaga gcagagttta
     2581 tgactttgct cttgtccagt ctagacatgg tgaattcgat atcaagctta tcgataccgt
     2641 ccgccgcagc gcagatggtc gcgcccgtgc ccccctatct cgcgcctcgc gtggtgcggt
     2701 ccggctgggc cggcggcggc gcggacgcga ccaaggtggc cgggaagggg agtttgcggg
     2761 ggaccggcga gtgacgtcag cgcgccttca gtgctgaggc ggcggtggcg cgcgccgcca
     2821 ggcgggggcg aaggcactgt ccgcggtgct gaagctggca gtgcgcacgc gcctcgccgc
     2881 atcctgtttc ccctccccct ctctgatagg ggatgcgcaa tttggggaat gggggttggg
     2941 tgcttgtcca gtgggtcggg gtcggtcgtc aggtaggcac ccccaccccg cctcatcctg
     3001 gtcctaaaac ccacttgcac tcatacgcag ggccctctgc agtatcaagt gccacctgac
     3061 gtctccctat cagtgataga gaagtcgaca cgtctcgagc tccctatcag tgatagagaa
     3121 ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc gacacgtctc gagctcccta
     3181 tcagtgatag agaaggtacg tctagaacgt ctccctatca gtgatagaga agtcgacacg
     3241 tctcgagctc cctatcagtg atagagaagg tacgtctaga acgtctccct atcagtgata
     3301 gagaagtcga cacgtctcga gctccctatc agtgatagag aagctacccc ctatataagc
     3361 agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
     3421 catagaagac accgggaccg atccagcctg gatccggtac cggatcctct agagtcgact
     3481 ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta gctggattgt
     3541 agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat tatacgaagt
     3601 tatggcgcgc ctcacttgtc caaggaattc ttactttgat tatcgataag cttgacctgg
     3661 ttcggggtag aacgttactc agtaaaccag tcctactgaa gaattcttac ttgcccagtc
     3721 tgttcatctc gatgtcggtg tagctgccgc ccagctcgtc catgcccatc acatcggtaa
     3781 aggccttttg ccactccttg aagttgagct cggtcttgga gtgcttgagc tccgtcttac
     3841 ggaacacgta catcggctgg ttcttcagat agttagccgc cattggcttg gcaaaggtgt
     3901 aggtggtccg cgcagtgctc cggtagcgct tgccatttcc agtggtgtaa ctccacttaa
     3961 aggtactgat gatggttttg tcgttggggt aagtcttctt cgacctgcac cagtccgcag
     4021 cggtcagcga gttggtcatc acaggaccgt cagcagggaa accagtcccc ttcacctggg
     4081 cctctccttt gatgtggctt ccctcgtagg tgtagcggta gttaacagta agggaggcac
     4141 catcttcaaa ctgcattgtg cgatggactt ggtatccgga gccatctacc atggcggcct
     4201 ggaaaggcga catcccgtca gggtagggca ggtactgatg gaagccatac ccgatatgag
     4261 ggaccagaat ccagggggag aactggaggt cacccttggt ggacttcagg tttaactcct
     4321 cataaccatc atttggattg ccggtgccct gacccaccat gtcaaagtcc acaccgttga
     4381 tggagccaaa gatgtgtaac tcatgtgtcg ctgggagaga ggccatgtta tcctcctcgc
     4441 ccttgctcac catggtggcg ctagcataac ttcgtataaa gtatcctata cgaagttatt
     4501 tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa taacttcgta
     4561 taatgtatgc tatacgaagt tatgaattcg atatcaagct tatcgataat caacctctgg
     4621 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     4681 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     4741 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     4801 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     4861 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     4921 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     4981 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca
     5041 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     5101 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     5161 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga
     5221 gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc
     5281 cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
     5341 ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg
     5401 gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca
     5461 atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc
     5521 cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta
     5581 gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta
     5641 cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt
     5701 ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     5761 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     5821 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     5881 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5941 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6001 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     6061 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6121 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     6181 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     6241 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     6301 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     6361 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     6421 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     6481 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     6541 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     6601 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     6661 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6721 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6781 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6841 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6901 ctga
//
