LOCUS       sequence-329443-        6823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cre-inactivated GFP-2A-Tetanus toxin light chain under 
            control of the TRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6823)
  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 6823)
  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 (Cre-OFF)
FEATURES             Location/Qualifiers
     source          1..6823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   531..671
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   542..671
                     /label=AAV2 ITR
     protein_bind    join(828..846,847..869,870..888,889..911,912..930,931..953,
                     954..972,973..995,996..1014,1015..1037,1038..1056,
                     1057..1079,1080..1098)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1131..1169
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(1304..1337)
                     /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(1348..1381)
                     /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             join(1448..1450,1451..1453,1454..2164)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1448..1450,1451..1453,1454..2164)
                     /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(1493..1514)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1754..1773)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2101..2122
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2186..2251
                     /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"
     protein_bind    3667..3700
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3711..3744
                     /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    3768..4356
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3821..3841)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4239..4250)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4391..4408)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4397..4604
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4627..4756
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4818..5406)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4898..4917)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5577..6368,6369..6437))
                     /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     6200..6219
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6438..6542)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6610..6628
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6666..6688)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6788..6807
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca atagaccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga
      121 tagagttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg
      481 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc cctgcaggca
      541 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
      601 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      661 taggggttcc tgcggccgca cgcgaaacaa ttctgcagga attcggggcc gcggaggctg
      721 gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct ccaggcgatc
      781 tgacggttca ctaaacgagc tctgcttata taggtcgagt ttaccactcc ctatcagtga
      841 tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc
      901 gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat
      961 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg
     1021 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact
     1081 ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga ggtaggcgtg
     1141 tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
     1201 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc
     1261 ccgaattcga gctcggtacc cggggatcct ctagtcgacg tcgataactt cgtatagcat
     1321 acattatacg aagttatccg aatcgcaata acttcgtata ggatacttta tacgaagtta
     1381 tttggcgcgc caaatttaaa taacgcaacg taccttcgag ggggcccaag gaattcttac
     1441 ttgtacgatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     1501 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     1561 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     1621 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     1681 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     1741 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1801 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1861 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     1921 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1981 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2041 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     2101 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     2161 caagggatcc aaaattgtcg ctcctgtcaa acaaactctt aactttgatt tactcaaact
     2221 ggctggggat gtagaaagca atccaggtcc agaattcatg ccgatcacca tcaacaactt
     2281 ccgttactcc gacccggtta acaacgacac catcatcatg atggaaccgc cgtactgcaa
     2341 aggtctagac atctactaca aagccttcaa aatcaccgac cgtatctgga tcgttccgga
     2401 acgttacgaa tttggtacca aaccggaaga cttcaacccg ccgtcctccc tgatcgaagg
     2461 tgcttccgaa tactacgacc cgaactacct gcgtaccgac tccgacaaag accgtttcct
     2521 gcagaccatg gttaaactgt tcaaccgtat caaaaacaac gttgctggtg aagctctgct
     2581 ggacaaaatc atcaacgcta tcccgtacct gggtaactcc tactccctgc tggacaaatt
     2641 cgacaccaac tccaactccg tttccttcaa cctgctggaa caggatccgt ccggtgctac
     2701 caccaaatcc gctatgctga ccaacctgat catcttcggt ccgggtccgg ttctgaacaa
     2761 aaacgaagtt cgtggtatcg ttctgcgtgt tgacaacaaa aactacttcc cgtgccgtga
     2821 cggtttcggt tccatcatgc agatggcttt ctgcccggaa tacgtaccga ccttcgacaa
     2881 cgttatcgaa aacatcacct ccctgaccat cggtaaatcc aaatacttcc aggacccggc
     2941 tctgctgctg atgcacgaac tgatccacgt tctgcacggt ctgtacggca tgcaggtttc
     3001 ctcccacgaa atcatcccgt ccaaacagga aatctacatg cagcacacct acccgatctc
     3061 cgctgaagag ctcttcacct tcggtggtca ggacgctaac ctgatctcca tcgatatcaa
     3121 aaacgacctg tacgaaaaaa ccctgaacga ctacaaagct atcgctaaca aactgtccca
     3181 ggttacctcc tgcaacgacc cgaacatcga catcgactcc tacaaacaga tctatcagca
     3241 gaagtatcag tttgataagg attccaacgg ccagtatata gtgaacgagg ataagtttca
     3301 gatactgtat aactccataa tgtatggctt tactgagata gagctgggca agaagtttaa
     3361 cataaagact cgcttaagct atttttccat gaaccacgat cctgtgaaga tacctaacct
     3421 gctggatgat actatatata acgatactga gggctttaac atagagtcca aggatctgaa
     3481 gtccgagtat aagggccaga acatgagggt gaacactaac gcctttagga acgtcgacgg
     3541 ttccggtctg gtttccaaac tgatcggtct gtgcaaaaaa atcatcccgc cgaccaacat
     3601 ccgtgaaaac ctgtacaacc gtaccgctta agctagctag ttggcgcgcc aacctggccg
     3661 gccttgataa cttcgtataa tgtatgctat acgaagttat ccgaatcgca ataacttcgt
     3721 ataaagtatc ctatacgaag ttatcgaaga tcatcaagct tatcgataat caacctctgg
     3781 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3841 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3901 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3961 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     4021 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     4081 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     4141 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca
     4201 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     4261 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     4321 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga
     4381 gatcgatctg cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4441 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4501 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4561 agcaaggggg aggattggga agacaatagc aggcatgctg gggacacgtg cggaccgagc
     4621 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4681 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggcgg cctcagtgag cgagcgagcg
     4741 cgcagctgcc tgcaggacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4801 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4861 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4921 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4981 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5041 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5101 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5161 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5221 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5281 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5341 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5401 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5461 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5521 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5581 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5641 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5701 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5761 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5821 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5881 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5941 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6001 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6061 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6121 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6181 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6241 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6301 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6361 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6421 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6481 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6541 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     6601 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     6661 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     6721 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     6781 aactatgcgg catcagagca gattgtactg agagtgcacc ata
//
