LOCUS       sequence-177105-        6328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To generate AAV virus that will express eNpHR-EYFP in Cre-dependent 
            recombination under control of tetO promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6328)
  AUTHORS   Lee D, Creed M, Jung K, Stefanelli T, Wendler DJ, Oh WC, Mignocchi 
            NL, Luscher C, Kwon HB
  TITLE     Temporally precise labeling and control of neuromodulatory circuits 
            in the mammalian brain.
  JOURNAL   Nat Methods. 2017 May;14(5):495-503. doi: 10.1038/nmeth.4234. Epub 
            2017 Apr 3.
  PUBMED    28369042
REFERENCE   2  (bases 1 to 6328)
  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"; doi: 
            "10.1038/nmeth.4234"; journalName: "Nat Methods"; date: "2017-05"; 
            volume: "14"; issue: "5"; pages: "495-503"
COMMENT     Sequence Label: pAAV-TRE-FLEX-eNpHR-EYFP
FEATURES             Location/Qualifiers
     source          1..6328
                     /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    join(298..316,317..339,340..358,359..381,382..400,401..423,
                     424..442,443..465,466..484,485..507,508..526,527..549,
                     550..568)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        601..639
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(778..811)
                     /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(862..895)
                     /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(931..1641,1642..1644))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(973..994)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1322..1341
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1581..1602
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2590..2623
                     /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    2674..2707
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2732..3320
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2785..2805)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3203..3214)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3355..3372)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3361..3568
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3591..3731
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3591..3720
                     /label=AAV2 ITR
     rep_origin      3806..4261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3893..3912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4103..4124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4278..4297)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4397..4419
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4457..4475)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4543..4647
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4648..4716,4717..5508)
                     /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(4866..4885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5679..6267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6168..6187
                     /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 cgcgaaacaa ttctgcagga attcggggcc
      181 gcggaggctg gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct
      241 ccaggcgatc tgacggttca ctaaacgagc tctgcttata taggtcgagt ttaccactcc
      301 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
      361 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
      421 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
      481 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
      541 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga
      601 ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc
      661 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc
      721 ctccgcggcc ccgaattcga gctcggtacc cggggatcct ctagagtcga ctccggaata
      781 acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt gtagctgcta
      841 ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa gttatggcgc
      901 gcctcattac acctcgttct cgtagcagaa cttgtacagc tcgtccatgc cgagagtgat
      961 cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt
     1021 gctcagggcg gactggtagc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc
     1081 gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg
     1141 gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt
     1201 gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
     1261 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg
     1321 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg
     1381 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggg cgaagcactg
     1441 caggccgtag ccgaaggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
     1501 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac
     1561 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     1621 gaacagctcc tcgcccttgc tcacggcggc cgcatcatca gccggggtcc cagaagcaga
     1681 tggaacatcc aaaatgctgc cggacacgac agattcgttt gacgtcagat agttcaggag
     1741 caagaatgcg aaaatgtact ttgccacgat atccaggaaa gaatatcccc agctcgtcac
     1801 cccaacgggc aacaccgcga taccctcgac tccaagagcc cacacaattg gataccccag
     1861 ccacatcact actgtcaaca gcttcagggt attgaacatg tcagcggttc ccgcggcttt
     1921 ggcgtcctgt gcccactcca ccagcaggat atacaagacc accagaaagc atgcacaact
     1981 gatagcgtac cagaaccatc tcatgaggtg ggaggaggta gtcagggccg cggcaaggcc
     2041 agtcacgcac atagcgatat cgaaagtgat agctgtgaag agctttgtag cattgcttcc
     2101 ggccaggaga cccagagcga ggagaatcat gggcgtggaa agtgcccagg tgagataccg
     2161 tccccacatg gtgactacac catctacctc ttctcctccc agcatcactg agctgccttc
     2221 tgcaaaatgg cccgctggca tttcaagaac ggaaattgtc aggccgctcg ccaatccagt
     2281 gtaggaggca atgctgacga caggcacaag gatggttgac acagcaataa gttttgcccg
     2341 tggatcatcg agtcctcggg tcataaaaac gaacagcagt atactcagtc ctgcaagtgc
     2401 gatgttgata tagagactgc ttgcaagcaa agggtcgttc agcacgaact cgaacaactc
     2461 cctttgggta acctcggctt gaagggccac ggcactctcg gtcacgggag gcagggtctc
     2521 tgtgtcctga agcagagaga acagagagac gacgaggagc aggggcgtac ccctcatggt
     2581 ggcgctagca taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa
     2641 ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg
     2701 aagttatgaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     2761 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     2821 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     2881 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     2941 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3001 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3061 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3121 ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg
     3181 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3241 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3301 cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcga tctgcctcga
     3361 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3421 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3481 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3541 gggaagacaa tagcaggcat gctggggaca cgtgcggacc gagcggccgc aggaacccct
     3601 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     3661 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     3721 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3781 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3841 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     3901 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3961 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4021 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4081 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4141 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4201 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     4261 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4321 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4381 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4441 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4501 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4561 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4621 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4681 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4741 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4801 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4861 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4921 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4981 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5041 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5101 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5161 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5221 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5281 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5341 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5401 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5461 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5521 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5581 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5641 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5701 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5761 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5821 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5881 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5941 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6001 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6061 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6121 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6181 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6241 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6301 gccttttgct ggccttttgc tcacatgt
//
