LOCUS       sequence-197132-        5539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tTA2 under the CAG promoter in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5539)
  AUTHORS   Sakaguchi R, Leiwe MN, Imai T
  TITLE     Bright multicolor labeling of neuronal circuits with fluorescent 
            proteins and chemical tags.
  JOURNAL   eLife. 2018 Nov 20;7. pii: 40350. doi: 10.7554/eLife.40350.
  PUBMED    30454553
REFERENCE   2  (bases 1 to 5539)
  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: "2018-11-20"; volume: "7. pii: 40350. doi"; pages: " 
            10.7554/eLife.40350"
COMMENT     Sequence Label: pCAG-tTA
FEATURES             Location/Qualifiers
     source          1..5539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1731..2348,2349..2354,2355..2390,2391..2393,2394..2429,
                     2430..2432,2433..2468,2469..2477)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(2563..2582)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2628..2683
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2682..2701)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3042..3058)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3042..3058)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3055..3077)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3066..3082
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3090..3096,3097..3114,3115..3120))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3135..3156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3215..3410
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3261..3396
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3416..3550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3453..3472)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3507..3526
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3618..3635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3789..4377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3869..3888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4548..5339,5340..5408))
                     /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     5171..5190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5409..5513)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcgccacc atgtctagac
     1741 tggacaagag caaagtcata aactctgctc tggaattact caatgaagtc ggtatcgaag
     1801 gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc ctgtactggc
     1861 acgtgaagaa caagcgggcc ctgctcgatg ccctggcaat cgagatgctg gacaggcatc
     1921 atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg aacaacgcca
     1981 agtcattccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat ctcggcaccc
     2041 gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag
     2101 gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt acactgggct
     2161 gcgtattgga ggatcaggag catcaagtag caaaagagga aagagagaca cctaccaccg
     2221 attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccatcag ggagccgaac
     2281 ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag ctaaagtgcg
     2341 aaagcggcgg gccggccgac gcccttgacg attttgactt agacatgctc ccagccgatg
     2401 cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat tttgaccttg
     2461 acatgctccc cgggtaagcg gccgcactcc tcaggtgcag gctgcctatc agaaggtggt
     2521 ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa
     2581 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     2641 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     2701 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     2761 catatgctgg ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc
     2821 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     2881 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     2941 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     3001 gaagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     3061 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3121 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3181 ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag
     3241 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3301 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     3361 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt
     3421 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3481 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     3541 tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3601 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3661 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3721 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3781 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3841 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3901 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3961 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     4021 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4081 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4141 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4201 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4261 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4321 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4381 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4441 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4501 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4561 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4621 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4681 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4741 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4801 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4861 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4921 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4981 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5041 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5101 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5161 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5221 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5281 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5341 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5401 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5461 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5521 ccccgaaaag tgccacctg
//
