LOCUS       sequence-486989-        6762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-ETAG in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6762)
  AUTHORS   E11 Bio
  TITLE     E11 Bio PRISM Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6762)
  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            "Unpublished"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-ETAG
FEATURES             Location/Qualifiers
     source          1..6762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(97..116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      910..1498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1399..1418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1560..1689
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1699..1820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1736..1755)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1790..1809
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1844..1852,1853..1888,1889..1891,1892..1927,
                     1928..1930,1931..1966,1967..1972,1973..2590))
                     /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    3046..3064
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3083..3101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3120..3138
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3157..3175
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3197..3221
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3320..3353)
                     /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(3404..3437)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4029..4038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4290..4299
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4306..4339
                     /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    4390..4423
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4448..5036
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4501..4521)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4919..4930)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5068..5544
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5267..5286)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5584..5724
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5584..5713
                     /label=AAV2 ITR
     rep_origin      5799..6254
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5886..5905)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6096..6117
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6271..6290)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6390..6412
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6450..6468)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6536..6640
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6641..6709,6710..6762,1..739)
                     /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 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
       61 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      121 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      421 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
      781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
      841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
      901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
      961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1021 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc
     1561 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg
     1621 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
     1681 ggggttcctg cggccgcaaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     1741 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     1801 aaactcatca atgtatctta tcatgtctga gatccttact tagttacccg gggagcatgt
     1861 caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc aaggtcaaag tcgtcaaggg
     1921 catcggctgg gagcatgtct aagtcaaaat cgtcaagggc gtcggccggc ccgccgcttt
     1981 cgcactttag ctgtttctcc aggccacata tgattagttc caggccgaaa aggaaggcag
     2041 gttcggctcc ctgatggtcg aacagctcaa ttgcttgtct cagaagtggg ggcatagaat
     2101 cggtggtagg tgtctctctt tcctcttttg ctacttgatg ctcctgatcc tccaatacgc
     2161 agcccagtgt aaagtggccc acggcggaca gagcgtacag tgcgttctcc agggagaagc
     2221 cttgctgaca caggaacgcg agctgatttt ccagggtttc gtactgtttc tctgttgggc
     2281 gggtgccgag atgcacttta gccccgtcgc gatgtgagag gagagcacag cggaatgact
     2341 tggcgttgtt ccgcagaaag tcttgccatg actcgccttc cagggggcag aagtgggtat
     2401 gatgcctgtc cagcatctcg attgccaggg catcgagcag ggcccgcttg ttcttcacgt
     2461 gccagtacag ggtaggctgc tcaactccca gcttttgagc gagtttcctt gtcgtcaggc
     2521 cttcgatacc gacttcattg agtaattcca gagcagagtt tatgactttg ctcttgtcca
     2581 gtctagacat ggtgaattcg atatcaagct tatcgatacc gtccgccgca gcgcagatgg
     2641 tcgcgcccgt gcccccctat ctcgcgcctc gcgtggtgcg gtccggctgg gccggcggcg
     2701 gcgcggacgc gaccaaggtg gccgggaagg ggagtttgcg ggggaccggc gagtgacgtc
     2761 agcgcgcctt cagtgctgag gcggcggtgg cgcgcgccgc caggcggggg cgaaggcact
     2821 gtccgcggtg ctgaagctgg cagtgcgcac gcgcctcgcc gcatcctgtt tcccctcccc
     2881 ctctctgata ggggatgcgc aatttgggga atgggggttg ggtgcttgtc cagtgggtcg
     2941 gggtcggtcg tcaggtaggc acccccaccc cgcctcatcc tggtcctaaa acccacttgc
     3001 actcatacgc agggccctct gcagtatcaa gtgccacctg acgtctccct atcagtgata
     3061 gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc tagaacgtct
     3121 ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat agagaagcta
     3181 ccccctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
     3241 gctgttttga cctccataga agacaccggg accgatccag cctggatccg gtaccggatc
     3301 ctctagagtc gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat
     3361 ggtagctgga ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat
     3421 acattatacg aagttatggc gcgcctcact tgtccaagga attcttactt tgattatcga
     3481 taagcttgac ctggaggaac atgacccata cagcaagcta ccagtcctac tgaagaattc
     3541 ttatcttggc tcaagagggt caggatatgg aacaggagca ccacttccgc ccagctcgtc
     3601 catgcccatc acatcggtaa aggccttttg ccactccttg aagttgagct cggtcttgga
     3661 gtgcttgagc tccgtcttac ggaacacgta catcggctgg ttcttcagat agttagccgc
     3721 cattggcttg gcaaaggtgt aggtggtccg cgcagtgctc cggtagcgct tgccatttcc
     3781 agtggtgtaa ctccacttaa aggtactgat gatggttttg tcgttggggt aagtcttctt
     3841 cgacctgcac cagtccgcag cggtcagcga gttggtcatc acaggaccgt cagcagggaa
     3901 accagtcccc ttcacctggg cctctccttt gatgtggctt ccctcgtagg tgtagcggta
     3961 gttaacagta agggaggcac catcttcaaa ctgcattgtg cgatggactt ggtatccgga
     4021 gccatctacc atggcggcct ggaaaggcga catcccgtca gggtagggca ggtactgatg
     4081 gaagccatac ccgatatgag ggaccagaat ccagggggag aactggaggt cacccttggt
     4141 ggacttcagg tttaactcct cataaccatc atttggattg ccggtgccct gacccaccat
     4201 gtcaaagtcc acaccgttga tggagccaaa gatgtgtaac tcatgtgtcg ctgggagaga
     4261 ggccatgtta tcctcctcgc ccttgctcac catggtggcg ctagcataac ttcgtataaa
     4321 gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg
     4381 gtgcaaagaa taacttcgta taatgtatgc tatacgaagt tatgaattcg atatcaagct
     4441 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4501 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4561 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     4621 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     4681 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     4741 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     4801 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     4861 gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     4921 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     4981 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     5041 ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct
     5101 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt
     5161 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta
     5221 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca
     5281 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg
     5341 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc
     5401 taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac
     5461 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga
     5521 accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc
     5581 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     5641 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     5701 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     5761 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     5821 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     5881 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5941 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6001 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6061 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6121 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     6181 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     6241 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6301 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     6361 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     6421 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     6481 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     6541 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6601 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     6661 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6721 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cg
//
