LOCUS       sequence-486972-        6916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-MoonTag in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6916)
  AUTHORS   E11 Bio
  TITLE     E11 Bio PRISM Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6916)
  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            "Unpublished"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-MoonTag
FEATURES             Location/Qualifiers
     source          1..6916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(104..123)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      917..1505
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1406..1425
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1567..1696
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1706..1827
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1743..1762)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1797..1816
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1851..1859,1860..1895,1896..1898,1899..1934,
                     1935..1937,1938..1973,1974..1979,1980..2597))
                     /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    3053..3071
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3090..3108
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3127..3145
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3164..3182
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3201..3219
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3238..3256
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3275..3293
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3312..3330
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3352..3376
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3475..3508)
                     /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(3559..3592)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3699..3743)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     regulatory      4190..4199
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4451..4460
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4467..4500
                     /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    4551..4584
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4609..5197
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4662..4682)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5080..5091)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5229..5705
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5428..5447)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5745..5885
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5745..5874
                     /label=AAV2 ITR
     rep_origin      5960..6415
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6047..6066)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6257..6278
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6432..6451)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6551..6573
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6611..6629)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6697..6801
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6802..6870,6871..6916,1..746)
                     /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 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
       61 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      121 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      181 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      241 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      301 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      361 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      421 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      481 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      541 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      601 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      661 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      721 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
      781 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
      841 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
      901 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      961 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1021 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1081 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1141 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1201 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1261 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1321 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1381 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1441 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1501 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1561 acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg
     1621 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
     1681 atcactaggg gttcctgcgg ccgcaaactt gtttattgca gcttataatg gttacaaata
     1741 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     1801 tttgtccaaa ctcatcaatg tatcttatca tgtctgagat ccttacttag ttacccgggg
     1861 agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca gcatatcaag gtcaaagtcg
     1921 tcaagggcat cggctgggag catgtctaag tcaaaatcgt caagggcgtc ggccggcccg
     1981 ccgctttcgc actttagctg tttctccagg ccacatatga ttagttccag gccgaaaagg
     2041 aaggcaggtt cggctccctg atggtcgaac agctcaattg cttgtctcag aagtgggggc
     2101 atagaatcgg tggtaggtgt ctctctttcc tcttttgcta cttgatgctc ctgatcctcc
     2161 aatacgcagc ccagtgtaaa gtggcccacg gcggacagag cgtacagtgc gttctccagg
     2221 gagaagcctt gctgacacag gaacgcgagc tgattttcca gggtttcgta ctgtttctct
     2281 gttgggcggg tgccgagatg cactttagcc ccgtcgcgat gtgagaggag agcacagcgg
     2341 aatgacttgg cgttgttccg cagaaagtct tgccatgact cgccttccag ggggcagaag
     2401 tgggtatgat gcctgtccag catctcgatt gccagggcat cgagcagggc ccgcttgttc
     2461 ttcacgtgcc agtacagggt aggctgctca actcccagct tttgagcgag tttccttgtc
     2521 gtcaggcctt cgataccgac ttcattgagt aattccagag cagagtttat gactttgctc
     2581 ttgtccagtc tagacatggt gaattcgata tcaagcttat cgataccgtc cgccgcagcg
     2641 cagatggtcg cgcccgtgcc cccctatctc gcgcctcgcg tggtgcggtc cggctgggcc
     2701 ggcggcggcg cggacgcgac caaggtggcc gggaagggga gtttgcgggg gaccggcgag
     2761 tgacgtcagc gcgccttcag tgctgaggcg gcggtggcgc gcgccgccag gcgggggcga
     2821 aggcactgtc cgcggtgctg aagctggcag tgcgcacgcg cctcgccgca tcctgtttcc
     2881 cctccccctc tctgataggg gatgcgcaat ttggggaatg ggggttgggt gcttgtccag
     2941 tgggtcgggg tcggtcgtca ggtaggcacc cccaccccgc ctcatcctgg tcctaaaacc
     3001 cacttgcact catacgcagg gccctctgca gtatcaagtg ccacctgacg tctccctatc
     3061 agtgatagag aagtcgacac gtctcgagct ccctatcagt gatagagaag gtacgtctag
     3121 aacgtctccc tatcagtgat agagaagtcg acacgtctcg agctccctat cagtgataga
     3181 gaaggtacgt ctagaacgtc tccctatcag tgatagagaa gtcgacacgt ctcgagctcc
     3241 ctatcagtga tagagaaggt acgtctagaa cgtctcccta tcagtgatag agaagtcgac
     3301 acgtctcgag ctccctatca gtgatagaga agctaccccc tatataagca gagctcgttt
     3361 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca
     3421 ccgggaccga tccagcctgg atccggtacc ggatcctcta gagtcgactc cggaataact
     3481 tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta gctgctatta
     3541 gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt atggcgcgcc
     3601 tcacttgtcc aaggaattct tactttgatt atcgataagc ttgacctggt tcatgtgtga
     3661 acggctccaa acgaaccagt cctactgaag aattcttaca ggctggccca cttgtccagc
     3721 tccagcagct cctgctcgtt cttgctgccg cccagctcgt ccatgcccat cacatcggta
     3781 aaggcctttt gccactcctt gaagttgagc tcggtcttgg agtgcttgag ctccgtctta
     3841 cggaacacgt acatcggctg gttcttcaga tagttagccg ccattggctt ggcaaaggtg
     3901 taggtggtcc gcgcagtgct ccggtagcgc ttgccatttc cagtggtgta actccactta
     3961 aaggtactga tgatggtttt gtcgttgggg taagtcttct tcgacctgca ccagtccgca
     4021 gcggtcagcg agttggtcat cacaggaccg tcagcaggga aaccagtccc cttcacctgg
     4081 gcctctcctt tgatgtggct tccctcgtag gtgtagcggt agttaacagt aagggaggca
     4141 ccatcttcaa actgcattgt gcgatggact tggtatccgg agccatctac catggcggcc
     4201 tggaaaggcg acatcccgtc agggtagggc aggtactgat ggaagccata cccgatatga
     4261 gggaccagaa tccaggggga gaactggagg tcacccttgg tggacttcag gtttaactcc
     4321 tcataaccat catttggatt gccggtgccc tgacccacca tgtcaaagtc cacaccgttg
     4381 atggagccaa agatgtgtaa ctcatgtgtc gctgggagag aggccatgtt atcctcctcg
     4441 cccttgctca ccatggtggc gctagcataa cttcgtataa agtatcctat acgaagttat
     4501 ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt
     4561 ataatgtatg ctatacgaag ttatgaattc gatatcaagc ttatcgataa tcaacctctg
     4621 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     4681 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     4741 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     4801 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     4861 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     4921 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     4981 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc
     5041 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     5101 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     5161 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag
     5221 agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg
     5281 ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact
     5341 aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg
     5401 ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac
     5461 aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc
     5521 ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt
     5581 agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct
     5641 acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg
     5701 tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     5761 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     5821 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     5881 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     5941 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     6001 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     6061 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     6121 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     6181 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     6241 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     6301 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     6361 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     6421 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     6481 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     6541 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     6601 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     6661 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6721 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6781 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6841 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6901 gctgaagatc agttgg
//
