LOCUS       sequence-486993-        6907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bidirectional AAV transfer plasmid expressing hSyn-tTA and 
            TRE-FLEX-mNeonGreen-ALFA 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  JOURNAL   Unpublished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            "Unpublished"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-ALFA
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(106..125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      919..1507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1408..1427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1569..1698
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    1708..1829
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1745..1764)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1799..1818
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1853..1861,1862..1897,1898..1900,1901..1936,
                     1937..1939,1940..1975,1976..1981,1982..2599))
                     /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    3055..3073
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3092..3110
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3129..3147
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3166..3184
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3203..3221
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3240..3258
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3277..3295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3314..3332
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3354..3378
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3477..3510)
                     /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(3561..3594)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3701..3739)
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     regulatory      4183..4192
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4444..4453
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4460..4493
                     /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    4544..4577
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4602..5190
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4655..4675)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5073..5084)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5222..5698
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5421..5440)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5738..5878
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5738..5867
                     /label=AAV2 ITR
     rep_origin      5953..6408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6040..6059)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6250..6271
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6425..6444)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6544..6566
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6604..6622)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6690..6794
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6795..6863,6864..6907,1..748)
                     /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 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
       61 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      121 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
      181 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
      241 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
      301 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
      361 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
      421 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
      481 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
      541 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
      601 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
      661 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
      721 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
      781 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
      841 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
      901 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
      961 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1021 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     1081 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1141 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1201 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1261 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1321 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1381 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1441 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1501 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1561 tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg
     1621 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
     1681 ccatcactag gggttcctgc ggccgcaaac ttgtttattg cagcttataa tggttacaaa
     1741 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     1801 ggtttgtcca aactcatcaa tgtatcttat catgtctgag atccttactt agttacccgg
     1861 ggagcatgtc aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt
     1921 cgtcaagggc atcggctggg agcatgtcta agtcaaaatc gtcaagggcg tcggccggcc
     1981 cgccgctttc gcactttagc tgtttctcca ggccacatat gattagttcc aggccgaaaa
     2041 ggaaggcagg ttcggctccc tgatggtcga acagctcaat tgcttgtctc agaagtgggg
     2101 gcatagaatc ggtggtaggt gtctctcttt cctcttttgc tacttgatgc tcctgatcct
     2161 ccaatacgca gcccagtgta aagtggccca cggcggacag agcgtacagt gcgttctcca
     2221 gggagaagcc ttgctgacac aggaacgcga gctgattttc cagggtttcg tactgtttct
     2281 ctgttgggcg ggtgccgaga tgcactttag ccccgtcgcg atgtgagagg agagcacagc
     2341 ggaatgactt ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc agggggcaga
     2401 agtgggtatg atgcctgtcc agcatctcga ttgccagggc atcgagcagg gcccgcttgt
     2461 tcttcacgtg ccagtacagg gtaggctgct caactcccag cttttgagcg agtttccttg
     2521 tcgtcaggcc ttcgataccg acttcattga gtaattccag agcagagttt atgactttgc
     2581 tcttgtccag tctagacatg gtgaattcga tatcaagctt atcgataccg tccgccgcag
     2641 cgcagatggt cgcgcccgtg cccccctatc tcgcgcctcg cgtggtgcgg tccggctggg
     2701 ccggcggcgg cgcggacgcg accaaggtgg ccgggaaggg gagtttgcgg gggaccggcg
     2761 agtgacgtca gcgcgccttc agtgctgagg cggcggtggc gcgcgccgcc aggcgggggc
     2821 gaaggcactg tccgcggtgc tgaagctggc agtgcgcacg cgcctcgccg catcctgttt
     2881 cccctccccc tctctgatag gggatgcgca atttggggaa tgggggttgg gtgcttgtcc
     2941 agtgggtcgg ggtcggtcgt caggtaggca cccccacccc gcctcatcct ggtcctaaaa
     3001 cccacttgca ctcatacgca gggccctctg cagtatcaag tgccacctga cgtctcccta
     3061 tcagtgatag agaagtcgac acgtctcgag ctccctatca gtgatagaga aggtacgtct
     3121 agaacgtctc cctatcagtg atagagaagt cgacacgtct cgagctccct atcagtgata
     3181 gagaaggtac gtctagaacg tctccctatc agtgatagag aagtcgacac gtctcgagct
     3241 ccctatcagt gatagagaag gtacgtctag aacgtctccc tatcagtgat agagaagtcg
     3301 acacgtctcg agctccctat cagtgataga gaagctaccc cctatataag cagagctcgt
     3361 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
     3421 caccgggacc gatccagcct ggatccggta ccggatcctc tagagtcgac tccggaataa
     3481 cttcgtatag gatactttat acgaagttat gcagaatggt agctggattg tagctgctat
     3541 tagcaatatg aaacctctta ataacttcgt atagcataca ttatacgaag ttatggcgcg
     3601 cctcacttgt ccaaggaatt cttactttga ttatcgataa gcttgacctg ggcgtaaaga
     3661 tatggggmaa ctaacaacca gtcctactga agaattctta ctcggtcagt cttcttctca
     3721 gctcctcctc cagtctgctg ccgcccagct cgtccatgcc catcacatcg gtaaaggcct
     3781 tttgccactc cttgaagttg agctcggtct tggagtgctt gagctccgtc ttacggaaca
     3841 cgtacatcgg ctggttcttc agatagttag ccgccattgg cttggcaaag gtgtaggtgg
     3901 tccgcgcagt gctccggtag cgcttgccat ttccagtggt gtaactccac ttaaaggtac
     3961 tgatgatggt tttgtcgttg gggtaagtct tcttcgacct gcaccagtcc gcagcggtca
     4021 gcgagttggt catcacagga ccgtcagcag ggaaaccagt ccccttcacc tgggcctctc
     4081 ctttgatgtg gcttccctcg taggtgtagc ggtagttaac agtaagggag gcaccatctt
     4141 caaactgcat tgtgcgatgg acttggtatc cggagccatc taccatggcg gcctggaaag
     4201 gcgacatccc gtcagggtag ggcaggtact gatggaagcc atacccgata tgagggacca
     4261 gaatccaggg ggagaactgg aggtcaccct tggtggactt caggtttaac tcctcataac
     4321 catcatttgg attgccggtg ccctgaccca ccatgtcaaa gtccacaccg ttgatggagc
     4381 caaagatgtg taactcatgt gtcgctggga gagaggccat gttatcctcc tcgcccttgc
     4441 tcaccatggt ggcgctagca taacttcgta taaagtatcc tatacgaagt tatttgcctt
     4501 aacccagaaa ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt
     4561 atgctatacg aagttatgaa ttcgatatca agcttatcga taatcaacct ctggattaca
     4621 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     4681 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     4741 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     4801 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     4861 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     4921 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     4981 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga
     5041 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     5101 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     5161 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta
     5221 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     5281 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     5341 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     5401 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     5461 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     5521 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     5581 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     5641 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     5701 gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     5761 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     5821 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     5881 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     5941 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     6001 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     6061 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     6121 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     6181 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     6241 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     6301 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     6361 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     6421 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     6481 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     6541 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     6601 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     6661 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6721 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     6781 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     6841 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     6901 atcagtt
//
