LOCUS       sequence-329447-        6965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cre-dependent GFP-2A-synaptophysinmRuby under control of the
            TRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6965)
  AUTHORS   Sabatini PV, Wang J, Rupp AC, Affinati AH, Flak JN, Li C, Olson DP, 
            Myers MG Jr
  TITLE     tTARGIT AAVs mediate the sensitive and flexible manipulation of 
            intersectional neuronal populations in mice.
  JOURNAL   eLife. 2021 Mar 11;10. pii: 66835. doi: 10.7554/eLife.66835.
  PUBMED    33704065
REFERENCE   2  (bases 1 to 6965)
  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: "eLife."; 
            date: "2021-03-11"; volume: "10. pii: 66835. doi"; pages: " 
            10.7554/eLife.66835"
COMMENT     Sequence Label: pAAV-TRE-DIO-GFP-2A-SynmRuby
FEATURES             Location/Qualifiers
     source          1..6965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    join(27..45,46..68,69..87,88..110,111..129,130..152,
                     153..171,172..194,195..213,214..236,237..255,256..278,
                     279..297)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        330..368
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(503..536)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(547..580)
                     /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)."
     CDS             complement(593..1273)
                     /codon_start=1
                     /product="monomeric variant of the red fluorescent protein 
                     eqFP611 (Kredel et al., 2009)"
                     /label=mRuby
                     /translation="MNSLIKENMRMKVVLEGSVNGHQFKCTGEGEGNPYMGTQTMRIKV
                     IEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGVITV
                     MQDTSLEDGCLVYHAQVRGVNFPSNGAVMQKKTKGWEPNTEMMYPADGGLRGYTHMALK
                     VDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFA
                     GLGGG"
     CDS             complement(2225..2278)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(join(2279..2989,2990..2992,2993..2995))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2279..2989,2990..2992,2993..2995))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2321..2342)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2670..2689
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2929..2950
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3008..3041
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3052..3085
                     /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)."
     misc_feature    3109..3697
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3162..3182)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3580..3591)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3732..3749)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3738..3945
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3968..4097
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4159..4747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4239..4258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4918..5709,5710..5778))
                     /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     5541..5560
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5779..5883)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5951..5969
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6007..6029)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6129..6148
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6165..6620)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6302..6323)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6514..6533
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6695..6835
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6706..6835
                     /label=AAV2 ITR
ORIGIN
        1 ctgcttatat aggtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag
       61 tttaccactc cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag
      121 tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa
      181 gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc
      241 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa
      301 gtgaaagtcg agctcggtac ccgggtcgag gtaggcgtgt acggtgggag gcctatataa
      361 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      421 tccatagaag acaccgggac cgatccagcc tccgcggccc cgaattcgag ctcggtaccc
      481 ggggatcctc tagtcgacgt cgataacttc gtatagcata cattatacga agttatccga
      541 atcgcaataa cttcgtatag gatactttat acgaagttat ttggcgcgcc taccctccgc
      601 ccaggccggc gaacttggcc acggcgtgct cccgctgcac cacgaacatc tcgttgtcgg
      661 actcttccag cctttccagc cggtggtcca cggcgtggat gccgggcatc ttgatgttgc
      721 ccacggtttt cttggatcgg taggtggtca cgaagctgca gctcaggtgc cctccgccgt
      781 ccaccttcag ggccatgtgg gtgtagcctc tcagtccgcc gtcagcgggg tacatcatct
      841 cggtgttggg ctcccagccc ttggttttct tctgcatcac ggcgccgttg ctgggaaaat
      901 tcacgcccct gacctgggca tggtacacca ggcagccatc ttccaggctg gtgtcctgca
      961 tcacggtgat cacgccgccg tcctcgtatc tggtcactct ctcccaggtg aagccctcgg
     1021 ggaagctctg cttgaagaaa tcggggatgc ccttggggta cttgatgaag gtccggctgc
     1081 cgtacatgaa ggatgtggcc aggatgtcga aggcgaaggg cagaggtccg ccctcgatca
     1141 ctttgatccg catggtctgg gtgcccatgt aggggttgcc ctcgccctcg ccggtgcact
     1201 tgaactggtg gccgttcacg ctgccttcca gcaccacctt catccgcatg ttttctttga
     1261 tcaggctgtt catcgtacgg gtggcgaccg gtggatccga catctgattg gagaaggagg
     1321 tgggcgcacc ctgttggccg tagccttgct gcccatagtc gccctgaggc ccgtagccac
     1381 cgccaccgcc gctggctggc tgcccgtaat cgggttgata accaccctga ggcccgtagg
     1441 agtcctgggg cccatagcct ccggggccct gcccatagcc cgcatcgccg taggcatcgc
     1501 caggagctgg ttgcttttct ggggcgcctg gaggtgcgcg catgaatggg gcggcccagc
     1561 ctgtctcctt gaacacgaac cataggttgc caacccagag caccaggttc aggaagccaa
     1621 acaccaccga ggtgttgagt cctgaagtca cagggtccct cagttccttg catgtgtttc
     1681 ctgtctggcg gcacataggc atctccttga taatgttctc tgggtcagtg gccatcttca
     1741 catcggacag gcctttggcc caggcggatg agctaactag ccacatgaaa gcgaacactg
     1801 ctgtggccag gaagtccatc attggccctt tgttgttctc tcggtacttg ttctgcagga
     1861 agatgtaggt ggccagggcc cccatggagt agaggaaggc aaacacagcc acggtgacaa
     1921 agaattcagc cgaggaggag tagtcaccaa ctaggaagat cttggtagtg ccccctttaa
     1981 cgcaggaggg tgcatcaaag tacacttggt gcagcctgaa tgggtactca aattcgactt
     2041 cgatgttgag ggcactctcc gtcttgttgg cacactccac gctcagccga agctctccgg
     2101 tgtagctgcc gcacgtagca aaggcgaaga tggcaaagac ccactgcagc accttcacga
     2161 agccaagggg ctccttgacc acccggaact gacccccagc caccagctga ttcaccacgt
     2221 ccatggggcc ggggttctcc tccacgtcgc cgcaggtcag caggctgccc ctgccctcct
     2281 tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca
     2341 tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt
     2401 tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga
     2461 gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct
     2521 tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc
     2581 ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg
     2641 tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga
     2701 tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca
     2761 tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc acgagggtgg
     2821 gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg
     2881 tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca
     2941 gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc accatgggcc
     3001 ggccttgata acttcgtata atgtatgcta tacgaagtta tccgaatcgc aataacttcg
     3061 tataaagtat cctatacgaa gttatcgaag atcatcaagc ttatcgataa tcaacctctg
     3121 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3181 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3241 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3301 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3361 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3421 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3481 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc
     3541 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     3601 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     3661 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag
     3721 agatcgatct gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3781 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3841 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3901 cagcaagggg gaggattggg aagacaatag caggcatgct ggggacacgt gcggaccgag
     3961 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4021 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggcg gcctcagtga gcgagcgagc
     4081 gcgcagctgc ctgcaggaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4141 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4201 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4261 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4321 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4381 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4441 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4501 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4561 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4621 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4681 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4741 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4801 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4861 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4921 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4981 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5041 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5101 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5161 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5221 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5281 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5341 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5401 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5461 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5521 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5581 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5641 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5701 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5761 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5821 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5881 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     5941 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     6001 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     6061 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     6121 taactatgcg gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa
     6181 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac
     6241 cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt
     6301 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     6361 aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg
     6421 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     6481 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     6541 taaggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     6601 tgcgccgcta cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac
     6661 agatgcgtaa ggagaaaata ccgcatcagg cgcccctgca ggcagctgcg cgctcgctcg
     6721 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca
     6781 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc
     6841 cgcacgcgaa acaattctgc aggaattcgg ggccgcggag gctggatcgg tcccggtgtc
     6901 ttctatggag gtcaaaacag cgtggatggc gtctccaggc gatctgacgg ttcactaaac
     6961 gagct
//
