LOCUS       sequence-486966-        6907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-SPOT in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6907)
  AUTHORS   E11 Bio
  TITLE     E11 Bio PRISM Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6907)
  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            "Unpublished"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-SPOT
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(96..115)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      909..1497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1398..1417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1559..1688
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1698..1819
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1735..1754)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1789..1808
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1843..1851,1852..1887,1888..1890,1891..1926,
                     1927..1929,1930..1965,1966..1971,1972..2589))
                     /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    3045..3063
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3082..3100
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3119..3137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3156..3174
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3193..3211
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3230..3248
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3267..3285
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3304..3322
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3344..3368
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3467..3500)
                     /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(3551..3584)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3691..3726)
                     /codon_start=1
                     /product="engineered Spot-Tag(R) peptide that binds with 
                     high affinity to a nanobody"
                     /label=Spot-Tag
                     /translation="PDRVRAVSHWSS"
     regulatory      4173..4182
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4434..4443
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4450..4483
                     /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    4534..4567
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4592..5180
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4645..4665)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5063..5074)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5212..5688
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5411..5430)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5728..5868
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5728..5857
                     /label=AAV2 ITR
     rep_origin      5943..6398
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6030..6049)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6240..6261
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6415..6434)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6534..6556
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6594..6612)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6680..6784
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6785..6853,6854..6907,1..738)
                     /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 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
       61 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      121 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      181 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      241 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
      301 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
      361 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
      421 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
      481 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
      541 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
      601 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
      661 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
      721 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
      781 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
      841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
      901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
      961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1021 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     1081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     1321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     1381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     1501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
     1561 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg
     1621 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     1681 gggttcctgc ggccgcaaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     1741 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     1801 aactcatcaa tgtatcttat catgtctgag atccttactt agttacccgg ggagcatgtc
     1861 aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt cgtcaagggc
     1921 atcggctggg agcatgtcta agtcaaaatc gtcaagggcg tcggccggcc cgccgctttc
     1981 gcactttagc tgtttctcca ggccacatat gattagttcc aggccgaaaa ggaaggcagg
     2041 ttcggctccc tgatggtcga acagctcaat tgcttgtctc agaagtgggg gcatagaatc
     2101 ggtggtaggt gtctctcttt cctcttttgc tacttgatgc tcctgatcct ccaatacgca
     2161 gcccagtgta aagtggccca cggcggacag agcgtacagt gcgttctcca gggagaagcc
     2221 ttgctgacac aggaacgcga gctgattttc cagggtttcg tactgtttct ctgttgggcg
     2281 ggtgccgaga tgcactttag ccccgtcgcg atgtgagagg agagcacagc ggaatgactt
     2341 ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc agggggcaga agtgggtatg
     2401 atgcctgtcc agcatctcga ttgccagggc atcgagcagg gcccgcttgt tcttcacgtg
     2461 ccagtacagg gtaggctgct caactcccag cttttgagcg agtttccttg tcgtcaggcc
     2521 ttcgataccg acttcattga gtaattccag agcagagttt atgactttgc tcttgtccag
     2581 tctagacatg gtgaattcga tatcaagctt atcgataccg tccgccgcag cgcagatggt
     2641 cgcgcccgtg cccccctatc tcgcgcctcg cgtggtgcgg tccggctggg ccggcggcgg
     2701 cgcggacgcg accaaggtgg ccgggaaggg gagtttgcgg gggaccggcg agtgacgtca
     2761 gcgcgccttc agtgctgagg cggcggtggc gcgcgccgcc aggcgggggc gaaggcactg
     2821 tccgcggtgc tgaagctggc agtgcgcacg cgcctcgccg catcctgttt cccctccccc
     2881 tctctgatag gggatgcgca atttggggaa tgggggttgg gtgcttgtcc agtgggtcgg
     2941 ggtcggtcgt caggtaggca cccccacccc gcctcatcct ggtcctaaaa cccacttgca
     3001 ctcatacgca gggccctctg cagtatcaag tgccacctga cgtctcccta tcagtgatag
     3061 agaagtcgac acgtctcgag ctccctatca gtgatagaga aggtacgtct agaacgtctc
     3121 cctatcagtg atagagaagt cgacacgtct cgagctccct atcagtgata gagaaggtac
     3181 gtctagaacg tctccctatc agtgatagag aagtcgacac gtctcgagct ccctatcagt
     3241 gatagagaag gtacgtctag aacgtctccc tatcagtgat agagaagtcg acacgtctcg
     3301 agctccctat cagtgataga gaagctaccc cctatataag cagagctcgt ttagtgaacc
     3361 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc
     3421 gatccagcct ggatccggta ccggatcctc tagagtcgac tccggaataa cttcgtatag
     3481 gatactttat acgaagttat gcagaatggt agctggattg tagctgctat tagcaatatg
     3541 aaacctctta ataacttcgt atagcataca ttatacgaag ttatggcgcg cctcacttgt
     3601 ccaaggaatt cttactttga ttatcgataa gcttgacctg gtaggaggtg ggattatata
     3661 aaccacacca gtcctactga agaattctta gctgctccag tggctcacgg ctctcactct
     3721 gtcagggctg ccgcccagct cgtccatgcc catcacatcg gtaaaggcct tttgccactc
     3781 cttgaagttg agctcggtct tggagtgctt gagctccgtc ttacggaaca cgtacatcgg
     3841 ctggttcttc agatagttag ccgccattgg cttggcaaag gtgtaggtgg tccgcgcagt
     3901 gctccggtag cgcttgccat ttccagtggt gtaactccac ttaaaggtac tgatgatggt
     3961 tttgtcgttg gggtaagtct tcttcgacct gcaccagtcc gcagcggtca gcgagttggt
     4021 catcacagga ccgtcagcag ggaaaccagt ccccttcacc tgggcctctc ctttgatgtg
     4081 gcttccctcg taggtgtagc ggtagttaac agtaagggag gcaccatctt caaactgcat
     4141 tgtgcgatgg acttggtatc cggagccatc taccatggcg gcctggaaag gcgacatccc
     4201 gtcagggtag ggcaggtact gatggaagcc atacccgata tgagggacca gaatccaggg
     4261 ggagaactgg aggtcaccct tggtggactt caggtttaac tcctcataac catcatttgg
     4321 attgccggtg ccctgaccca ccatgtcaaa gtccacaccg ttgatggagc caaagatgtg
     4381 taactcatgt gtcgctggga gagaggccat gttatcctcc tcgcccttgc tcaccatggt
     4441 ggcgctagca taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa
     4501 ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg
     4561 aagttatgaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     4621 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     4681 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     4741 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     4801 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     4861 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     4921 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     4981 ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg
     5041 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     5101 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     5161 cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat
     5221 ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc
     5281 agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat
     5341 attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg
     5401 gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
     5461 atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc
     5521 caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
     5581 atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc
     5641 aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag
     5701 gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct
     5761 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     5821 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     5881 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     5941 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6001 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6061 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     6121 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     6181 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     6241 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     6301 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     6361 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     6421 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     6481 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     6541 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     6601 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     6661 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6721 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6781 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6841 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6901 tgcacga
//
