LOCUS       sequence-491312-        7384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of a green fluorescent protein biosensor to
            determine mitochondrial cellular reduction-oxidation (redox) 
            states..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7384)
  AUTHORS   Tubert C, Zampese E, Pancani T, Tkatch T, Surmeier DJ
  TITLE     Feed-forward metabotropic signaling by Cav1 Ca(2+) channels supports
            pacemaking in pedunculopontine cholinergic neurons.
  JOURNAL   Neurobiol Dis. 2023 Nov;188:106328. doi: 10.1016/j.nbd.2023.106328. 
            Epub 2023 Oct 16.
  PUBMED    37852390
REFERENCE   2  (bases 1 to 7384)
  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: "Neurobiol 
            Dis."; date: "2023-11"; volume: "188:106328. doi"; pages: " 
            10.1016/j.nbd.2023.106328. Epub 2023 Oct 16"
COMMENT     Sequence Label: pFB-CMV-DIO-MTS-roGFP-WPRE
FEATURES             Location/Qualifiers
     source          1..7384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        194..497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        498..701
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     652..672
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     698..722
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(803..836)
                     /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(887..920)
                     /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(932..1645,1646..1648,1649..1651))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(977..998)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1326..1345
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1585..1606
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    1712..1745
                     /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    1796..1829
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1854..2442
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1907..1927)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2325..2336)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2468..2487)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2469..2487)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2513..2647
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2676..2816
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2676..2805
                     /label=AAV2 ITR
     polyA_signal    2983..3117
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     mobile_element  3146..3311
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      3495..3949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3582..3601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3791..3812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3975..4079
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4080..4148,4149..4940)
                     /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(4298..4317)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5111..5699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5600..5619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  6004..6228
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(6295..6828)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7017..7022,7023..7039,7040..7045))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtagtaat caattacggg gtcattagtt catagcccat
      181 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      241 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      301 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      361 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      421 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      481 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      541 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      601 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      661 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
      721 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      781 gcctccaacc gtcgactccg gaataacttc gtataggata ctttatacga agttatgcag
      841 aatggtagct ggattgtagc tgctattagc aatatgaaac ctcttaataa cttcgtatag
      901 catacattat acgaagttat ggcgcgcctt attacttgta cagctcgtcc atgccgagag
      961 tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt
     1021 ctttgctcag ggcggagcag gtgctcaggt agtggttgtc gggcagcagc acggggccgt
     1081 cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt
     1141 tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga
     1201 cgttgtggca gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga
     1261 agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg
     1321 cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt
     1381 cgggcatggc ggacttgaag aagtcgtgcc gcttcatgtg gtcggggtag cggctgaagc
     1441 actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg
     1501 tggtgctgat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg
     1561 acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc
     1621 cggtgaacag ctcctcgccc ttgctcacca tgatgccgct gcgcttgaag aagcggatgc
     1681 tctggcgcag gctcagcatg gtggcgctag cataacttcg tataaagtat cctatacgaa
     1741 gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac
     1801 ttcgtataat gtatgctata cgaagttatg aattcgatat caagcttatc gataatcaac
     1861 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     1921 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     1981 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     2041 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     2101 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     2161 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     2221 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg
     2281 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     2341 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     2401 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgtacc
     2461 caattcgccc tatagtgagt cgtattacgc gcgcagcggc cgaccatggc ccaacttgtt
     2521 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2581 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     2641 ctggatctcc ggacacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     2701 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     2761 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggtctg
     2821 agacaataac cctgataaat gcttcaataa tgtaagcttg tcgagaagta ctagaggatc
     2881 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     2941 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     3001 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3061 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctga
     3121 tcactgatat cgcctaggag atccgaacca gataagtgaa atctagttcc aaactatttt
     3181 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca
     3241 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt
     3301 gtccgcccac agcggggcat ttttcttcct gttatgtttt taatcaaaca tcctgccaac
     3361 tccatgtgac aaaccgtcat cttcggctac tttttctctg tcacagaatg aaaatttttc
     3421 tgtcatctct tcgttattaa tgtttgtaat tgactgaata tcaacgctta tttgcagcct
     3481 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3541 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3601 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3661 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3721 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3781 gttcttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3841 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3901 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3961 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4021 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4081 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4141 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4201 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4261 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4321 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4381 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4441 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4501 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4561 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4621 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4681 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4741 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4801 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4861 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4921 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4981 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5041 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5101 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5161 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5221 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5281 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5341 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5401 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5461 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5521 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5581 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5641 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5701 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5761 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5821 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5881 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagacc
     5941 agccgcgtaa cctggcaaaa tcggttacgg ttgagtaata aatggatgcc ctgcgtaagc
     6001 gggtgtgggc ggacaataaa gtcttaaact gaacaaaata gatctaaact atgacaataa
     6061 agtcttaaac tagacagaat agttgtaaac tgaaatcagt ccagttatgc tgtgaaaaag
     6121 catactggac ttttgttatg gctaaagcaa actcttcatt ttctgaagtg caaattgccc
     6181 gtcgtattaa agaggggcgt ggccaagggc atggtaaaga ctatattcgc ggcgttgtga
     6241 caatttaccg aacaactccg cggccgggaa gccgatctcg gcttgaacga attgttaggt
     6301 ggcggtactt gggtcgatat caaagtgcat cacttcttcc cgtatgccca actttgtata
     6361 gagagccact gcgggatcgt caccgtaatc tgcttgcacg tagatcacat aagcaccaag
     6421 cgcgttggcc tcatgcttga ggagattgat gagcgcggtg gcaatgccct gcctccggtg
     6481 ctcgccggag actgcgagat catagatata gatctcacta cgcggctgct caaacttggg
     6541 cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat
     6601 gaatgtctta ctacggagca agttcccgag gtaatcggag tccggctgat gttgggagta
     6661 ggtggctacg tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac
     6721 ttggtcaggg ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt
     6781 tttagggcga ctgccctgct gcgtaacatc gttgctgctg cgtaacatcg ttgctgctcc
     6841 ataacatcaa acatcgaccc acggcgtaac gcgcttgctg cttggatgcc cgaggcatag
     6901 actgtacaaa aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc
     6961 tgcgttcggt caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagtt
     7021 tacgaaccga acaggcttat gtcaactggg ttcgtgcctt catccgtttc cacggtgtgc
     7081 gtcacccggc aaccttgggc agcagcgaag tcgaggcatt tctgtcctgg ctggcgaacg
     7141 agcgcaaggt ttcggtctcc acgcatcgtc aggcattggc ggccttgctg ttcttctacg
     7201 gcaaggtgct gtgcacggat ctgccctggc ttcaggagat cggaagacct cggccgtcgc
     7261 ggcgcttgcc ggtggtgctg accccggatg aagtggttcg catcctcggt tttctggaag
     7321 gcgagcatcg tttgttcgcc caggactcta gctatagttc tagtggttgg ctacagcttg
     7381 catg
//
