LOCUS       sequence-37037-e        5126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5126)
  AUTHORS   Hung YP, Albeck JG, Tantama M, Yellen G
  TITLE     Imaging Cytosolic NADH-NAD(+) Redox State with a Genetically Encoded
            Fluorescent Biosensor.
  JOURNAL   Cell Metab. 2011 Oct 5;14(4):545-54.
  PUBMED    21982714
REFERENCE   2  (bases 1 to 5126)
  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: "Cell 
            Metab."; date: "2011-10-5"; volume: "14(4)"; pages: "545-54"
COMMENT     Sequence Label: pRsetB-His7tag-Peredox-mCherry
FEATURES             Location/Qualifiers
     source          1..5126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..60
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        41..59
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             91..113
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             127..147
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             join(160..774,775..795,796..1530,1531..1533,1534..2163,
                     2164..2208)
                     /codon_start=1
                     /product="genetically encoded fluorescent biosensor of the 
                     cytosolic NADH-NAD+ redox state (Hung et al., 2011)"
                     /label=Peredox
                     /note="consists of two pH-insensitive mutant copies of the 
                     Thermus aquaticus redox-sensing repressor Rex (T-Rex) 
                     separated by circularly permuted monomeric T-Sapphire 
                     (cpmTS), a pH-resistant GFP variant"
                     /translation="MKVPEAAISRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQV
                     DKDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGES
                     FELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADLLVAA
                     GIKGILNFAPVVLEVPKEVAVENVDILAGLTRLSFAILNPTWSAAGGHGFTAHNVYIMA
                     DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSKLSKDPNE
                     KRDHMVLLEFVTAAGITHGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKF
                     SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVMVFARYPDHMKQHDFFKS
                     AMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNT
                     KVPEAAISRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGV
                     GYTVPVLKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVG
                     RPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPVVLEV
                     PKEVAVENVDFLAGLTRLSFAILNPKWREEMMGSGTGGNASDGGGSGG"
     CDS             2209..2919
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2354..2372)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2622..2641
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3003..3024
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3187..3291
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3292..3360,3361..4152)
                     /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(3510..3529)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4323..4911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4812..4831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5065..5082
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttggccgatt cattaatgca ggatctcgat cccgcgaaat taatacgact cactataggg
       61 agaccacaac ggtttccctc tagaaataat tttgtttaac tttaagaagg agatatacat
      121 atgaaacatc accatcatca tcatcatggt ggagctagca tgaaagttcc tgaagcagcc
      181 atttccagac tgattactta cctgagaatc ctggaagagc tggaagccca aggtgttcac
      241 cgcaccgcct ccgagcaact gggcgagctg gcccaggtca ccgccttcca ggttgataag
      301 gacctgtcct acttcggctc ctacggcact gacggcgttg gctacactgt accggtcctc
      361 aagagagaac tgcgccatat cctcggtctg aaccgcaaat ggggcctgtg catcgtgggg
      421 atgggccgcc tgggatccgc tcttgctgat tggcctggtt ttggcgagag cttcgagctg
      481 cgcggtttct tcgatgttga cccaggtatg gttggtcggc cggttcgcgg tggtgttatc
      541 gaacacgttg atctgctgcc ccagcgcgta cctggtcgca tcgaaatcgc tctgctgacg
      601 gttccacgcg aggcagcaca gaaggctgcc gacctgctgg ttgccgctgg catcaagggc
      661 atcctgaact tcgctccagt tgtactggag gttcccaagg aagtggctgt tgagaacgtt
      721 gacatcctgg ccggtctgac gcgtctgagc tttgccattc tgaaccccac gtggagcgca
      781 gcaggtgggc atggtttcac tgctcacaac gtgtatatca tggctgacaa acagaagaac
      841 ggtatcaagg ccaacttcaa gatccgccac aacatcgaag acggcggcgt tcagctggct
      901 gatcactacc agcagaacac cccaatcggc gacggccctg ttctgctgcc tgacaaccac
      961 tacttaagca tccagtctaa actgtccaaa gacccaaacg agaagcgcga ccacatggtt
     1021 ctgctggaat ttgttaccgc cgctggcatc acccacggca tggacgaact gtacaagggc
     1081 ggcaccgggg gatctatggt ttccaaaggc gaggaactgt tcactggtgt tgttcctatc
     1141 ctggttgaac tggacggcga cgttaacggc cacaaattca gcgttagcgg ggaaggcgaa
     1201 ggagacgcta cctacggcaa gctgactctg aagttcatct gcaccactgg caagctgcct
     1261 gttccttggc ctaccctggt tactaccttc tcctatggtg ttatggtttt cgccagatat
     1321 cctgaccaca tgaagcagca cgacttcttc aagagcgcca tgcctgaggg ctacgttcag
     1381 gaacgcacca tctttttcaa ggacgacggc aactataaga cacgcgctga ggttaagttc
     1441 gagggcgaca ctctggttaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1501 aacatcctgg gccataagct tgaatataac accaaggtac ctgaagccgc catcagccgc
     1561 ctcatcactt acctccgcat cctggaggag ctcgaagccc aaggcgttca ccgcactgca
     1621 agcgagcagc tgggcgaact ggcacaggtt acagcattcc aggttgacga agatctgtcc
     1681 tatttcggtt cctacggcac cgacggtgtt ggctacacag tccctgtcct gaagcgcgag
     1741 ctgcgccaca tcctgggcct gaaccgcaag tggggcttat gcatcgttgg catgggccgc
     1801 ctcggtagtg cactggcaga ctggccgggc tttggtgaat ctttcgaact gcgcggcttc
     1861 ttcgacgttg atccaggcat ggttgggcgc cctgttcgcg gtggcgttat cgagcatgtt
     1921 gacctcctgc cacagcgcgt cccgggtcgc atcgagatcg ccctgctgac cgttcctcgc
     1981 gaagctgcac agaaggcagc cgacttacta gtcgccgctg gtatcaaggg aattctgaac
     2041 ttcgcccctg ttgttctgga ggttcctaag gaagttgctg ttgaaaacgt tgatttcctg
     2101 gccggcctga cccgcctgag cttcgcaatc ctgaatccta agtggcgcga agaaatgatg
     2161 ggttccggaa ctgggggcaa cgcctctgac ggtggtgggt ctggtggtat ggtgagcaag
     2221 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     2281 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     2341 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     2401 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     2461 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     2521 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     2581 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     2641 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     2701 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     2761 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     2821 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     2881 cgccactcca ccggcggcat ggacgaactg tacaagtgac tcgaggggcc caagctgtag
     2941 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3001 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3061 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3121 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     3181 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3241 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3301 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3361 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3421 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3481 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3541 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3601 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3661 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3721 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3781 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3841 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3901 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3961 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4021 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4081 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4141 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4201 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4261 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4321 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4381 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4441 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4501 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4561 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4621 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4681 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4741 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4801 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4861 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4921 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4981 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5041 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     5101 atacgcaaac cgcctctccc cgcgcg
//
