LOCUS       sequence-314660-        6473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of nonbinding variant of inverse-response 
            citrate sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6473)
  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 6473)
  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-CitroffRH
FEATURES             Location/Qualifiers
     source          1..6473
                     /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"
     primer_bind     complement(973..994)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1234..1253)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1971..1992
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2064..2081)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2070..2294
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2340..2768
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2427..2446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2637..2658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2777..2797)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2782..3111
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2962..3097
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3024..3043
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3178..3972
                     /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(3232..3251)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3842..3861
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4146..4267
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4183..4202)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4237..4256
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4316..4332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4316..4332)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4329..4351)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4340..4356
                     /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(4364..4370,4371..4388,4389..4394))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4409..4430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4547..4564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4718..5306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4798..4817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5477..6268,6269..6337))
                     /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     6100..6119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6338..6442)
                     /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 ctcgagaatg gtgagcaagg gcgaggagct gttcaccggg
      961 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1021 ggcgagggtg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1081 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1141 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1201 ggctacatcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1261 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1321 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actggcagac cgaggagcgt
     1381 ctgcattatc aggtcgggca acgggcgctg attcaggcga tgcagatttc ggcgatgccg
     1441 gagctggtgg aggcagtgca gaaacgcgat ctcgccagaa tcaaagccct tatcggcccc
     1501 atgcgttcgt tctccgacgc cacctacatc accgtcggtg atgccagcgg ccaggccctc
     1561 tatgccgtca atcctgatga aatcggcaaa tcgatggaag gcggcgatag cgatgaggcg
     1621 ttgataaacg ctaaaagcta cgtgtcagtg cgcaaaggct cgctgggatc gtcgctgcgc
     1681 ggtaaatcgc cgattcagga cgcaaccggc aaggtgatcg gcattgtgtc ggtaggctat
     1741 accattgagc aactggaacc gaattttgtc tatatcatgg ccgacaagca gaagaacggc
     1801 atcaaggcga acttccacat ccgccacaac atcgaggacg gcggcgtgca gctcgcctac
     1861 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1921 ctgagcgtgc agtcccaact ttcgaaagac cccaacgaga agcgcgatca catggtcctg
     1981 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg tcgagctgta caagtaaaag
     2041 cttaagttta aaccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2101 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2161 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2221 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2281 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2341 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2401 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2461 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2521 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2581 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2641 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2701 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2761 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2821 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2881 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2941 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3001 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3061 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3121 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3181 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3241 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3301 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3361 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3421 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3481 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3541 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     3601 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     3661 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     3721 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     3781 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     3841 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     3901 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     3961 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4021 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4081 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4141 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4201 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4261 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4321 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4381 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4441 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4501 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4561 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4621 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4681 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4741 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4801 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4861 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4921 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4981 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5041 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5101 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5161 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5221 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5281 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5341 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5401 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5461 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5521 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5581 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5641 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5701 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5761 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5821 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5881 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5941 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6001 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6061 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6121 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6181 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6241 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6301 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6361 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6421 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
