LOCUS       sequence-486967-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-SunTag in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6928)
  AUTHORS   E11 Bio
  TITLE     E11 Bio PRISM Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6928)
  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            "Unpublished"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-SunTag
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(107..126)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      920..1508
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1409..1428
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1570..1699
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1709..1830
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1746..1765)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1800..1819
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1854..1862,1863..1898,1899..1901,1902..1937,
                     1938..1940,1941..1976,1977..1982,1983..2600))
                     /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    3056..3074
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3093..3111
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3130..3148
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3167..3185
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3204..3222
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3241..3259
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3278..3296
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3315..3333
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3355..3379
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3478..3511)
                     /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(3562..3595)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3702..3758)
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     regulatory      4205..4214
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4466..4475
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4482..4515
                     /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    4566..4599
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4624..5212
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4677..4697)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5095..5106)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5244..5720
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5443..5462)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5760..5900
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5760..5889
                     /label=AAV2 ITR
     rep_origin      5975..6430
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6062..6081)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6272..6293
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6447..6466)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6566..6588
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6626..6644)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6712..6816
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6817..6885,6886..6928,1..749)
                     /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 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
       61 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      121 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      181 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      241 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      301 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      361 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      421 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      481 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      541 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      601 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      661 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      721 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
      781 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
      841 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
      901 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      961 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1021 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1081 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1141 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1201 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1261 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1321 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1381 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1441 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1501 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1561 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
     1621 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
     1681 tccatcacta ggggttcctg cggccgcaaa cttgtttatt gcagcttata atggttacaa
     1741 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     1801 tggtttgtcc aaactcatca atgtatctta tcatgtctga gatccttact tagttacccg
     1861 gggagcatgt caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc aaggtcaaag
     1921 tcgtcaaggg catcggctgg gagcatgtct aagtcaaaat cgtcaagggc gtcggccggc
     1981 ccgccgcttt cgcactttag ctgtttctcc aggccacata tgattagttc caggccgaaa
     2041 aggaaggcag gttcggctcc ctgatggtcg aacagctcaa ttgcttgtct cagaagtggg
     2101 ggcatagaat cggtggtagg tgtctctctt tcctcttttg ctacttgatg ctcctgatcc
     2161 tccaatacgc agcccagtgt aaagtggccc acggcggaca gagcgtacag tgcgttctcc
     2221 agggagaagc cttgctgaca caggaacgcg agctgatttt ccagggtttc gtactgtttc
     2281 tctgttgggc gggtgccgag atgcacttta gccccgtcgc gatgtgagag gagagcacag
     2341 cggaatgact tggcgttgtt ccgcagaaag tcttgccatg actcgccttc cagggggcag
     2401 aagtgggtat gatgcctgtc cagcatctcg attgccaggg catcgagcag ggcccgcttg
     2461 ttcttcacgt gccagtacag ggtaggctgc tcaactccca gcttttgagc gagtttcctt
     2521 gtcgtcaggc cttcgatacc gacttcattg agtaattcca gagcagagtt tatgactttg
     2581 ctcttgtcca gtctagacat ggtgaattcg atatcaagct tatcgatacc gtccgccgca
     2641 gcgcagatgg tcgcgcccgt gcccccctat ctcgcgcctc gcgtggtgcg gtccggctgg
     2701 gccggcggcg gcgcggacgc gaccaaggtg gccgggaagg ggagtttgcg ggggaccggc
     2761 gagtgacgtc agcgcgcctt cagtgctgag gcggcggtgg cgcgcgccgc caggcggggg
     2821 cgaaggcact gtccgcggtg ctgaagctgg cagtgcgcac gcgcctcgcc gcatcctgtt
     2881 tcccctcccc ctctctgata ggggatgcgc aatttgggga atgggggttg ggtgcttgtc
     2941 cagtgggtcg gggtcggtcg tcaggtaggc acccccaccc cgcctcatcc tggtcctaaa
     3001 acccacttgc actcatacgc agggccctct gcagtatcaa gtgccacctg acgtctccct
     3061 atcagtgata gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc
     3121 tagaacgtct ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat
     3181 agagaaggta cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc
     3241 tccctatcag tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc
     3301 gacacgtctc gagctcccta tcagtgatag agaagctacc ccctatataa gcagagctcg
     3361 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag
     3421 acaccgggac cgatccagcc tggatccggt accggatcct ctagagtcga ctccggaata
     3481 acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt gtagctgcta
     3541 ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa gttatggcgc
     3601 gcctcacttg tccaaggaat tcttactttg attatcgata agcttgacct ggttgagtgc
     3661 attagagcct cctgcagacc agtcctactg aagaattctt acttcttcag tctggccacc
     3721 tcgttctcca ggtggtagtt cttgctcagc agctcctcgc tgccgcccag ctcgtccatg
     3781 cccatcacat cggtaaaggc cttttgccac tccttgaagt tgagctcggt cttggagtgc
     3841 ttgagctccg tcttacggaa cacgtacatc ggctggttct tcagatagtt agccgccatt
     3901 ggcttggcaa aggtgtaggt ggtccgcgca gtgctccggt agcgcttgcc atttccagtg
     3961 gtgtaactcc acttaaaggt actgatgatg gttttgtcgt tggggtaagt cttcttcgac
     4021 ctgcaccagt ccgcagcggt cagcgagttg gtcatcacag gaccgtcagc agggaaacca
     4081 gtccccttca cctgggcctc tcctttgatg tggcttccct cgtaggtgta gcggtagtta
     4141 acagtaaggg aggcaccatc ttcaaactgc attgtgcgat ggacttggta tccggagcca
     4201 tctaccatgg cggcctggaa aggcgacatc ccgtcagggt agggcaggta ctgatggaag
     4261 ccatacccga tatgagggac cagaatccag ggggagaact ggaggtcacc cttggtggac
     4321 ttcaggttta actcctcata accatcattt ggattgccgg tgccctgacc caccatgtca
     4381 aagtccacac cgttgatgga gccaaagatg tgtaactcat gtgtcgctgg gagagaggcc
     4441 atgttatcct cctcgccctt gctcaccatg gtggcgctag cataacttcg tataaagtat
     4501 cctatacgaa gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc
     4561 aaagaataac ttcgtataat gtatgctata cgaagttatg aattcgatat caagcttatc
     4621 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     4681 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     4741 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     4801 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     4861 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     4921 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     4981 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     5041 ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     5101 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     5161 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     5221 cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc
     5281 tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca
     5341 tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
     5401 gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct
     5461 ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc
     5521 tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat
     5581 ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct
     5641 aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
     5701 ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca
     5761 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     5821 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     5881 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5941 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     6001 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     6061 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     6121 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     6181 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     6241 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     6301 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     6361 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     6421 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6481 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     6541 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     6601 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     6661 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     6721 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     6781 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     6841 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6901 gtgaaagtaa aagatgctga agatcagt
//
