LOCUS       sequence-333909-        6767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cre-dependent eGFP-2A tetanus toxin light chain fragment 
            under control of the TRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6767)
  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 6767)
  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-eGFP-2A-TetanusToxin
FEATURES             Location/Qualifiers
     source          1..6767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    93..111
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    135..153
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    177..195
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    219..237
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    261..279
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    303..321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        354..392
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(527..560)
                     /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(571..604)
                     /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(2031..2096)
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             complement(join(2118..2828,2829..2831,2832..2834))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2118..2828,2829..2831,2832..2834))
                     /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(2160..2181)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2509..2528
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2768..2789
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2831..2840
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2876..2909
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2920..2953
                     /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    2977..3565
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3030..3050)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3448..3459)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3600..3617)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3606..3813
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3836..3965
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4027..4615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4107..4126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4786..5577,5578..5646))
                     /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     5409..5428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5647..5751)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5819..5837
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5875..5897)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5997..6016
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6033..6488)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6170..6191)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6382..6401
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6563..6703
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6574..6703
                     /label=AAV2 ITR
ORIGIN
        1 cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg gttcactaaa
       61 cgagctctgc ttatataggt cgagtttacc actccctatc agtgatagag aaaagtgaaa
      121 gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc
      181 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa
      241 gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc
      301 actccctatc agtgatagag aaaagtgaaa gtcgagctcg gtacccgggt cgaggtaggc
      361 gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
      421 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg
      481 gccccgaatt cgagctcggt acccggggat cctctagtcg acgtcgataa cttcgtatag
      541 catacattat acgaagttat ccgaatcgca ataacttcgt ataggatact ttatacgaag
      601 ttatttggcg cgccaaattt aaataacgca acgtaccttc gagggggccc ttaagcggta
      661 cggttgtaca ggttttcacg gatgttggtc ggcgggatga tttttttgca cagaccgatc
      721 agtttggaaa ccagaccgga accgtcgacg ttcctaaagg cgttagtgtt caccctcatg
      781 ttctggccct tatactcgga cttcagatcc ttggactcta tgttaaagcc ctcagtatcg
      841 ttatatatag tatcatccag caggttaggt atcttcacag gatcgtggtt catggaaaaa
      901 tagcttaagc gagtctttat gttaaacttc ttgcccagct ctatctcagt aaagccatac
      961 attatggagt tatacagtat ctgaaactta tcctcgttca ctatatactg gccgttggaa
     1021 tccttatcaa actgatactt ctgctgatag atctgtttgt aggagtcgat gtcgatgttc
     1081 gggtcgttgc aggaggtaac ctgggacagt ttgttagcga tagctttgta gtcgttcagg
     1141 gttttttcgt acaggtcgtt tttgatatcg atggagatca ggttagcgtc ctgaccaccg
     1201 aaggtgaaga gctcttcagc ggagatcggg taggtgtgct gcatgtagat ttcctgtttg
     1261 gacgggatga tttcgtggga ggaaacctgc atgccgtaca gaccgtgcag aacgtggatc
     1321 agttcgtgca tcagcagcag agccgggtcc tggaagtatt tggatttacc gatggtcagg
     1381 gaggtgatgt tttcgataac gttgtcgaag gtcggtacgt attccgggca gaaagccatc
     1441 tgcatgatgg aaccgaaacc gtcacggcac gggaagtagt ttttgttgtc aacacgcaga
     1501 acgataccac gaacttcgtt tttgttcaga accggacccg gaccgaagat gatcaggttg
     1561 gtcagcatag cggatttggt ggtagcaccg gacggatcct gttccagcag gttgaaggaa
     1621 acggagttgg agttggtgtc gaatttgtcc agcagggagt aggagttacc caggtacggg
     1681 atagcgttga tgattttgtc cagcagagct tcaccagcaa cgttgttttt gatacggttg
     1741 aacagtttaa ccatggtctg caggaaacgg tctttgtcgg agtcggtacg caggtagttc
     1801 gggtcgtagt attcggaagc accttcgatc agggaggacg gcgggttgaa gtcttccggt
     1861 ttggtaccaa attcgtaacg ttccggaacg atccagatac ggtcggtgat tttgaaggct
     1921 ttgtagtaga tgtctagacc tttgcagtac ggcggttcca tcatgatgat ggtgtcgttg
     1981 ttaaccgggt cggagtaacg gaagttgttg atggtgatcg gcatgaattc tggacctgga
     2041 ttgctttcta catccccagc cagtttgagt aaatcaaagt taagagtttg tttgacagga
     2101 gcgacaattt tggatccctt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
     2161 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac
     2221 tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
     2281 tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag
     2341 ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag
     2401 ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc
     2461 tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg
     2521 ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg
     2581 aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc
     2641 agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc
     2701 agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg
     2761 ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg
     2821 cccttgctca ccatggtggc gctagctagt tggcgcgcca acctggccgg ccttgataac
     2881 ttcgtataat gtatgctata cgaagttatc cgaatcgcaa taacttcgta taaagtatcc
     2941 tatacgaagt tatcgaagat catcaagctt atcgataatc aacctctgga ttacaaaatt
     3001 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3061 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3121 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     3181 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     3241 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     3301 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     3361 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg
     3421 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     3481 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     3541 atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag atcgatctgc
     3601 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3661 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3721 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3781 ggattgggaa gacaatagca ggcatgctgg ggacacgtgc ggaccgagcg gccgcaggaa
     3841 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     3901 cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     3961 gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4021 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4081 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4141 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4201 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4261 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4321 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4381 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4441 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4501 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4561 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4621 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4681 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4741 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4801 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4861 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4921 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4981 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5041 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5101 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5161 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5221 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5281 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5341 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5401 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5461 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5521 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5581 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5641 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5701 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5761 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     5821 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     5881 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     5941 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     6001 atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata ttttgttaaa
     6061 attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg aaatcggcaa
     6121 aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc cagtttggaa
     6181 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     6241 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
     6301 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     6361 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta aggcgctggc
     6421 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     6481 gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag atgcgtaagg
     6541 agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct cactgaggcc
     6601 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
     6661 gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg cacgcgaaac
     6721 aattctgcag gaattcgggg ccgcggaggc tggatcggtc ccggtgt
//
