LOCUS       sequence-196950-        5531 bp    DNA     circular SYN 14-APR-2025
DEFINITION  An AAV genome with tet-inducible, Cre-dependent expression of the 
            fluorescent protein eYFP with a C-terminal Hras farnesylation 
            sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5531)
  AUTHORS   Challis RC, Ravindra Kumar S, Chan KY, Challis C, Beadle K, Jang MJ,
            Kim HM, Rajendran PS, Tompkins JD, Shivkumar K, Deverman BE, 
            Gradinaru V
  TITLE     Systemic AAV vectors for widespread and targeted gene delivery in 
            rodents.
  JOURNAL   Nat Protoc. 2019 Feb;14(2):379-414. doi: 10.1038/s41596-018-0097-3.
  PUBMED    30626963
REFERENCE   2  (bases 1 to 5531)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Nat Protoc"; date: "2019-02"; volume: "14"; issue: "2"; pages: 
            "379-414"
COMMENT     Sequence Label: pAAV-TRE-DIO-eYFP-f
FEATURES             Location/Qualifiers
     source          1..5531
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    176..194
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    213..231
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    250..268
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    287..305
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    324..342
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    361..379
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    398..416
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    435..453
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     475..499
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(598..631)
                     /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(682..715)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(787..1497,1498..1500,1501..1503))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(829..850)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1178..1197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1437..1458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1500..1509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1516..1549
                     /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    1600..1633
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1658..2246
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1711..1731)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2129..2140)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2278..2754
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2477..2496)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2794..2934
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2794..2923
                     /label=AAV2 ITR
     rep_origin      3009..3464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3096..3115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3306..3327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3481..3500)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3600..3622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3660..3678)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3746..3850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3851..3919,3920..4711)
                     /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"
     primer_bind     complement(4069..4088)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4882..5470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5371..5390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtatcaa gtgccacctg acgtctccct
      181 atcagtgata gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc
      241 tagaacgtct ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat
      301 agagaaggta cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc
      361 tccctatcag tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc
      421 gacacgtctc gagctcccta tcagtgatag agaagctacc ccctatataa gcagagctcg
      481 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag
      541 acaccgggac cgatccagcc tggatccggt accggatcct ctagagtcga ctccggaata
      601 acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt gtagctgcta
      661 ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa gttatggcgc
      721 gccttaggag agcacacact tgcagctcat gcagccgggg ccactctcat caggagggtt
      781 cagcttcttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag
      841 caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact ggtagctcag
      901 gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg
      961 gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat
     1021 gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag
     1081 cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt
     1141 caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt
     1201 gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg
     1261 ctgcttcatg tggtcggggt agcgggcgaa gcactgcagg ccgtagccga aggtggtcac
     1321 gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt
     1381 gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc
     1441 gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac
     1501 catggtggcg ctagcataac ttcgtataaa gtatcctata cgaagttatt tgccttaacc
     1561 cagaaattat cactgttatt ctttagaatg gtgcaaagaa taacttcgta taatgtatgc
     1621 tatacgaagt tatgaattcg atatcaagct tatcgataat caacctctgg attacaaaat
     1681 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     1741 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     1801 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     1861 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     1921 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     1981 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     2041 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct
     2101 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     2161 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     2221 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg
     2281 tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg
     2341 cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc
     2401 tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc
     2461 tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc
     2521 actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg
     2581 ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt
     2641 ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg
     2701 cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt
     2761 ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     2821 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     2881 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     2941 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     3001 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3061 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3121 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3181 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     3241 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3301 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt
     3361 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3421 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     3481 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3541 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3601 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3661 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3721 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3781 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3841 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3901 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3961 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4021 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4081 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4141 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4201 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4261 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4321 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4381 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4441 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4501 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4561 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4621 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4681 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4741 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4801 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4861 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4921 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4981 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     5041 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5101 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5161 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5221 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5281 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5341 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5401 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5461 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5521 tgctcacatg t
//
