LOCUS       sequence-486969-        6907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-ProteinC in the other direction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6907)
  AUTHORS   E11 Bio
  TITLE     E11 Bio PRISM Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6907)
  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            "Unpublished"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-ProteinC
FEATURES             Location/Qualifiers
     source          1..6907
                     /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..3734)
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     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..6907,1..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 ttgacctggg gaagagttca
     3661 cctcacgact acccaccagt cctactgaag aattcttatt tgccgtcgat cagtctaggg
     3721 tccacctggt cctcgctgcc gcccagctcg 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 cagttgg
//
