LOCUS       sequence-309045-        6677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mitochondria-targeted nonbinding variant of inverse-response citrate
            sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6677)
  AUTHORS   Zhao Y, Shen Y, Wen Y, Campbell RE
  TITLE     High-Performance Intensiometric Direct- and Inverse-Response 
            Genetically Encoded Biosensors for Citrate.
  JOURNAL   ACS Cent Sci. 2020 Aug 26;6(8):1441-1450. doi: 
            10.1021/acscentsci.0c00518. Epub 2020 Jul 9.
  PUBMED    32875085
REFERENCE   2  (bases 1 to 6677)
  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"; doi: 
            "10.1021/acscentsci.0c00518"; journalName: "ACS Cent Sci"; date: 
            "2020-08-26- 26"; volume: "6"; issue: "8"; pages: "1441-1450"
COMMENT     Sequence Label: CMV-mito-CitroffRH
FEATURES             Location/Qualifiers
     source          1..6677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             928..1002
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1024..1098
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     primer_bind     complement(1177..1198)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1438..1457)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2175..2196
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2268..2285)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2274..2498
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2544..2972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2631..2650)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2841..2862
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2981..3001)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2986..3315
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3166..3301
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3228..3247
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3382..4176
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3436..3455)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4046..4065
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4350..4471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4387..4406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4441..4460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4520..4536)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4520..4536)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4533..4555)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4544..4560
                     /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(4568..4574,4575..4592,4593..4598))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4613..4634
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4751..4768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4922..5510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5002..5021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5681..6472,6473..6541))
                     /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     6304..6323
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6542..6646)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgagcatg tctgttctga ctcctctgct gctccggggt
      961 ctcacaggtt ccgcaagaag actccccgtg cctagggcca aaattcattc actgggggac
     1021 cccatgagcg tgctcacccc actcctgctg cgggggctga ccggcagcgc taggcggctg
     1081 ccagtcccca gggccaagat ccacagtctc ggcgatccca aggatccaac catggtgagc
     1141 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1201 aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta cggcaagctg
     1261 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1321 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1381 ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt cttcaaggac
     1441 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1501 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1561 tacaactggc agaccgagga gcgtctgcat tatcaggtcg ggcaacgggc gctgattcag
     1621 gcgatgcaga tttcggcgat gccggagctg gtggaggcag tgcagaaacg cgatctcgcc
     1681 agaatcaaag cccttatcgg ccccatgcgt tcgttctccg acgccaccta catcaccgtc
     1741 ggtgatgcca gcggccaggc cctctatgcc gtcaatcctg atgaaatcgg caaatcgatg
     1801 gaaggcggcg atagcgatga ggcgttgata aacgctaaaa gctacgtgtc agtgcgcaaa
     1861 ggctcgctgg gatcgtcgct gcgcggtaaa tcgccgattc aggacgcaac cggcaaggtg
     1921 atcggcattg tgtcggtagg ctataccatt gagcaactgg aaccgaattt tgtctatatc
     1981 atggccgaca agcagaagaa cggcatcaag gcgaacttcc acatccgcca caacatcgag
     2041 gacggcggcg tgcagctcgc ctaccactac cagcagaaca cccccatcgg cgacggcccc
     2101 gtgctgctgc ccgacaacca ctacctgagc gtgcagtccc aactttcgaa agaccccaac
     2161 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     2221 atggtcgagc tgtacaagta aaagcttaag tttaaaccgc tgatcagcct cgactgtgcc
     2281 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2341 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2401 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2461 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2521 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2581 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2641 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2701 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2761 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2821 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2881 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2941 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3001 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3061 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3121 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3181 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3241 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3301 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     3361 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3421 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3481 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3541 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3601 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3661 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3721 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3781 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3841 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3901 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3961 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4021 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4081 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4141 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4201 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4261 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4321 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4381 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4441 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4501 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4561 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4621 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4681 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4741 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4801 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4861 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4921 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4981 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5041 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5101 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5161 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5221 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5281 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5341 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5401 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5461 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5521 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5581 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5641 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5701 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5761 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5821 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5881 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5941 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6001 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6061 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6121 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6181 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6241 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6301 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6361 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6421 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6481 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6541 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6601 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6661 aagtgccacc tgacgtc
//
