LOCUS       sequence-486899-        6898 bp    DNA     circular SYN 23-SEP-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-NWS in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6898)
  AUTHORS   n/a
  TITLE     E11 Bio PRISM Collection Plasmids 2025
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6898)
  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            "unpublished"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-NWS
FEATURES             Location/Qualifiers
     source          1..6898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      272..281
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      533..542
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    549..582
                     /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    633..666
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    691..1279
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(744..764)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1162..1173)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    1311..1787
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1510..1529)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   1827..1967
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1827..1956
                     /label=AAV2 ITR
     rep_origin      2042..2497
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2129..2148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2339..2360
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2514..2533)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2633..2655
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2693..2711)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2779..2883
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2884..2952,2953..3744)
                     /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"
     primer_bind     complement(3102..3121)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3915..4503
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4404..4423
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   4565..4694
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    4704..4825
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4741..4760)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4795..4814
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4849..4857,4858..4893,4894..4896,4897..4932,
                     4933..4935,4936..4971,4972..4977,4978..5595))
                     /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    6051..6069
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6088..6106
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6125..6143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6162..6180
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6199..6217
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6236..6254
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6273..6291
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6310..6328
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6350..6374
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(6473..6506)
                     /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(6557..6590)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(6697..6720)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
ORIGIN
        1 ctccggtagc gcttgccatt tccagtggtg taactccact taaaggtact gatgatggtt
       61 ttgtcgttgg ggtaagtctt cttcgacctg caccagtccg cagcggtcag cgagttggtc
      121 atcacaggac cgtcagcagg gaaaccagtc cccttcacct gggcctctcc tttgatgtgg
      181 cttccctcgt aggtgtagcg gtagttaaca gtaagggagg caccatcttc aaactgcatt
      241 gtgcgatgga cttggtatcc ggagccatct accatggcgg cctggaaagg cgacatcccg
      301 tcagggtagg gcaggtactg atggaagcca tacccgatat gagggaccag aatccagggg
      361 gagaactgga ggtcaccctt ggtggacttc aggtttaact cctcataacc atcatttgga
      421 ttgccggtgc cctgacccac catgtcaaag tccacaccgt tgatggagcc aaagatgtgt
      481 aactcatgtg tcgctgggag agaggccatg ttatcctcct cgcccttgct caccatggtg
      541 gcgctagcat aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat
      601 tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga
      661 agttatgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
      721 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
      781 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
      841 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
      901 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
      961 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     1021 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     1081 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     1141 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     1201 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     1261 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     1321 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     1381 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     1441 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     1501 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     1561 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     1621 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     1681 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     1741 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     1801 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     1861 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     1921 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     1981 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     2041 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2101 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2161 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2221 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     2281 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2341 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     2401 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     2461 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     2521 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     2581 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     2641 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     2701 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     2761 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2821 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2881 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2941 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3001 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3061 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3121 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3181 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3241 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3301 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3361 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3421 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3481 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3541 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3601 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3661 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3721 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3781 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3841 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3901 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3961 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4021 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4081 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4141 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4201 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4261 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4321 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4381 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4441 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4501 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4561 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
     4621 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     4681 cactaggggt tcctgcggcc gcaaacttgt ttattgcagc ttataatggt tacaaataaa
     4741 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4801 tgtccaaact catcaatgta tcttatcatg tctgagatcc ttacttagtt acccggggag
     4861 catgtcaagg tcaaaatcgt caagagcgtc agcaggcagc atatcaaggt caaagtcgtc
     4921 aagggcatcg gctgggagca tgtctaagtc aaaatcgtca agggcgtcgg ccggcccgcc
     4981 gctttcgcac tttagctgtt tctccaggcc acatatgatt agttccaggc cgaaaaggaa
     5041 ggcaggttcg gctccctgat ggtcgaacag ctcaattgct tgtctcagaa gtgggggcat
     5101 agaatcggtg gtaggtgtct ctctttcctc ttttgctact tgatgctcct gatcctccaa
     5161 tacgcagccc agtgtaaagt ggcccacggc ggacagagcg tacagtgcgt tctccaggga
     5221 gaagccttgc tgacacagga acgcgagctg attttccagg gtttcgtact gtttctctgt
     5281 tgggcgggtg ccgagatgca ctttagcccc gtcgcgatgt gagaggagag cacagcggaa
     5341 tgacttggcg ttgttccgca gaaagtcttg ccatgactcg ccttccaggg ggcagaagtg
     5401 ggtatgatgc ctgtccagca tctcgattgc cagggcatcg agcagggccc gcttgttctt
     5461 cacgtgccag tacagggtag gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt
     5521 caggccttcg ataccgactt cattgagtaa ttccagagca gagtttatga ctttgctctt
     5581 gtccagtcta gacatggtga attcgatatc aagcttatcg ataccgtccg ccgcagcgca
     5641 gatggtcgcg cccgtgcccc cctatctcgc gcctcgcgtg gtgcggtccg gctgggccgg
     5701 cggcggcgcg gacgcgacca aggtggccgg gaaggggagt ttgcggggga ccggcgagtg
     5761 acgtcagcgc gccttcagtg ctgaggcggc ggtggcgcgc gccgccaggc gggggcgaag
     5821 gcactgtccg cggtgctgaa gctggcagtg cgcacgcgcc tcgccgcatc ctgtttcccc
     5881 tccccctctc tgatagggga tgcgcaattt ggggaatggg ggttgggtgc ttgtccagtg
     5941 ggtcggggtc ggtcgtcagg taggcacccc caccccgcct catcctggtc ctaaaaccca
     6001 cttgcactca tacgcagggc cctctgcagt atcaagtgcc acctgacgtc tccctatcag
     6061 tgatagagaa gtcgacacgt ctcgagctcc ctatcagtga tagagaaggt acgtctagaa
     6121 cgtctcccta tcagtgatag agaagtcgac acgtctcgag ctccctatca gtgatagaga
     6181 aggtacgtct agaacgtctc cctatcagtg atagagaagt cgacacgtct cgagctccct
     6241 atcagtgata gagaaggtac gtctagaacg tctccctatc agtgatagag aagtcgacac
     6301 gtctcgagct ccctatcagt gatagagaag ctacccccta tataagcaga gctcgtttag
     6361 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
     6421 gggaccgatc cagcctggat ccggtaccgg atcctctaga gtcgactccg gaataacttc
     6481 gtataggata ctttatacga agttatgcag aatggtagct ggattgtagc tgctattagc
     6541 aatatgaaac ctcttaataa cttcgtatag catacattat acgaagttat ggcgcgcctc
     6601 acttgtccaa ggaattctta ctttgattat cgataagctt gacctggtct aagacgcggg
     6661 ttggtacgga ataccagtcc tactgaagaa ttcttacttc tcgaactgag ggtggctcca
     6721 ggcgctgccg cccagctcgt ccatgcccat cacatcggta aaggcctttt gccactcctt
     6781 gaagttgagc tcggtcttgg agtgcttgag ctccgtctta cggaacacgt acatcggctg
     6841 gttcttcaga tagttagccg ccattggctt ggcaaaggtg taggtggtcc gcgcagtg
//
