LOCUS       sequence-486970-        6913 bp    DNA     circular SYN 23-SEP-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-OLLAS in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6913)
  AUTHORS   n/a
  TITLE     E11 Bio PRISM Collection Plasmids 2025
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6913)
  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            "unpublished"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-OLLAS
FEATURES             Location/Qualifiers
     source          1..6913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(98..117)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      911..1499
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1400..1419
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1561..1690
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1700..1821
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1737..1756)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1791..1810
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1845..1853,1854..1889,1890..1892,1893..1928,
                     1929..1931,1932..1967,1968..1973,1974..2591))
                     /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    3047..3065
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3084..3102
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3121..3139
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3158..3176
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3195..3213
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3232..3250
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3269..3287
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3306..3324
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3346..3370
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3469..3502)
                     /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(3553..3586)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3696..3737)
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     regulatory      4181..4190
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4442..4451
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4458..4491
                     /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    4542..4575
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4600..5188
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4653..4673)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5071..5082)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5220..5696
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5419..5438)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5736..5876
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5736..5865
                     /label=AAV2 ITR
     rep_origin      5951..6406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6038..6057)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6248..6269
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6423..6442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6542..6564
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6602..6620)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6688..6792
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6793..6861,6862..6913,1..740)
                     /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 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
       61 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
      121 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
      181 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
      241 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
      301 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
      361 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
      421 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
      481 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
      541 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
      601 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
      661 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
      721 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
      781 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
      841 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
      901 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
      961 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1021 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     1081 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1141 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1201 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1261 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     1321 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1381 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     1441 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     1501 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     1561 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
     1621 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
     1681 aggggttcct gcggccgcaa acttgtttat tgcagcttat aatggttaca aataaagcaa
     1741 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1801 caaactcatc aatgtatctt atcatgtctg agatccttac ttagttaccc ggggagcatg
     1861 tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa gtcgtcaagg
     1921 gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggccgg cccgccgctt
     1981 tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa aaggaaggca
     2041 ggttcggctc cctgatggtc gaacagctca attgcttgtc tcagaagtgg gggcatagaa
     2101 tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgatc ctccaatacg
     2161 cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc cagggagaag
     2221 ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt ctctgttggg
     2281 cgggtgccga gatgcacttt agccccgtcg cgatgtgaga ggagagcaca gcggaatgac
     2341 ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca gaagtgggta
     2401 tgatgcctgt ccagcatctc gattgccagg gcatcgagca gggcccgctt gttcttcacg
     2461 tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct tgtcgtcagg
     2521 ccttcgatac cgacttcatt gagtaattcc agagcagagt ttatgacttt gctcttgtcc
     2581 agtctagaca tggtgaattc gatatcaagc ttatcgatac cgtccgccgc agcgcagatg
     2641 gtcgcgcccg tgccccccta tctcgcgcct cgcgtggtgc ggtccggctg ggccggcggc
     2701 ggcgcggacg cgaccaaggt ggccgggaag gggagtttgc gggggaccgg cgagtgacgt
     2761 cagcgcgcct tcagtgctga ggcggcggtg gcgcgcgccg ccaggcgggg gcgaaggcac
     2821 tgtccgcggt gctgaagctg gcagtgcgca cgcgcctcgc cgcatcctgt ttcccctccc
     2881 cctctctgat aggggatgcg caatttgggg aatgggggtt gggtgcttgt ccagtgggtc
     2941 ggggtcggtc gtcaggtagg cacccccacc ccgcctcatc ctggtcctaa aacccacttg
     3001 cactcatacg cagggccctc tgcagtatca agtgccacct gacgtctccc tatcagtgat
     3061 agagaagtcg acacgtctcg agctccctat cagtgataga gaaggtacgt ctagaacgtc
     3121 tccctatcag tgatagagaa gtcgacacgt ctcgagctcc ctatcagtga tagagaaggt
     3181 acgtctagaa cgtctcccta tcagtgatag agaagtcgac acgtctcgag ctccctatca
     3241 gtgatagaga aggtacgtct agaacgtctc cctatcagtg atagagaagt cgacacgtct
     3301 cgagctccct atcagtgata gagaagctac cccctatata agcagagctc gtttagtgaa
     3361 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga
     3421 ccgatccagc ctggatccgg taccggatcc tctagagtcg actccggaat aacttcgtat
     3481 aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct attagcaata
     3541 tgaaacctct taataacttc gtatagcata cattatacga agttatggcg cgcctcactt
     3601 gtccaaggaa ttcttacttt gattatcgat aagcttgacc tggtcgtatc aaaacaggca
     3661 acatcgtcac cagtcctact gaagaattct tagctctttc ccatcagtct aggtcccagc
     3721 tcgttggcga atccgctgcc tcccagctcg tccatgccca tcacatcggt aaaggccttt
     3781 tgccactcct tgaagttgag ctcggtcttg gagtgcttga gctccgtctt acggaacacg
     3841 tacatcggct ggttcttcag atagttagcc gccattggct tggcaaaggt gtaggtggtc
     3901 cgcgcagtgc tccggtagcg cttgccattt ccagtggtgt aactccactt aaaggtactg
     3961 atgatggttt tgtcgttggg gtaagtcttc ttcgacctgc accagtccgc agcggtcagc
     4021 gagttggtca tcacaggacc gtcagcaggg aaaccagtcc ccttcacctg ggcctctcct
     4081 ttgatgtggc ttccctcgta ggtgtagcgg tagttaacag taagggaggc accatcttca
     4141 aactgcattg tgcgatggac ttggtatccg gagccatcta ccatggcggc ctggaaaggc
     4201 gacatcccgt cagggtaggg caggtactga tggaagccat acccgatatg agggaccaga
     4261 atccaggggg agaactggag gtcacccttg gtggacttca ggtttaactc ctcataacca
     4321 tcatttggat tgccggtgcc ctgacccacc atgtcaaagt ccacaccgtt gatggagcca
     4381 aagatgtgta actcatgtgt cgctgggaga gaggccatgt tatcctcctc gcccttgctc
     4441 accatggtgg cgctagcata acttcgtata aagtatccta tacgaagtta tttgccttaa
     4501 cccagaaatt atcactgtta ttctttagaa tggtgcaaag aataacttcg tataatgtat
     4561 gctatacgaa gttatgaatt cgatatcaag cttatcgata atcaacctct ggattacaaa
     4621 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     4681 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     4741 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     4801 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     4861 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     4921 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     4981 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc cacctggatt
     5041 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     5101 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     5161 cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatctacg
     5221 ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag
     5281 tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
     5341 tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
     5401 cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc
     5461 tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt
     5521 tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg
     5581 gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt
     5641 ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga
     5701 ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     5761 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     5821 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     5881 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     5941 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     6001 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     6061 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     6121 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     6181 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     6241 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     6301 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     6361 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     6421 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     6481 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     6541 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     6601 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     6661 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     6721 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     6781 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6841 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6901 cagttgggtg cac
//
