LOCUS       sequence-243129-        7466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative-control mutant for RAB-EKARev biosensor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7466)
  AUTHORS   Mehta S, Zhang Y, Roth RH, Zhang JF, Mo A, Tenner B, Huganir RL, 
            Zhang J
  TITLE     Single-fluorophore biosensors for sensitive and multiplexed 
            detection of signalling activities.
  JOURNAL   Nat Cell Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 
            10.1038/s41556-018-0200-6.
  PUBMED    30250062
REFERENCE   2  (bases 1 to 7466)
  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: "Nat Cell 
            Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 
            10.1038/s41556-018-0200-6."
COMMENT     Sequence Label: pcDNA3-RAB-EKARev(T/A)
FEATURES             Location/Qualifiers
     source          1..7466
                     /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"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1873..2220
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     promoter        complement(3019..3037)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3020..3037)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3057..3074)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3063..3287
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3333..3761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3420..3439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3630..3651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3770..3790)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3775..4104
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3955..4090
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4017..4036
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4171..4965
                     /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(4225..4244)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4835..4854
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5139..5260
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5176..5195)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5230..5249
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5309..5325)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5309..5325)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5322..5344)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5333..5349
                     /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(5357..5363,5364..5381,5382..5387))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5402..5423
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5540..5557)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5711..6299)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5791..5810)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6470..7261,7262..7330))
                     /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     7093..7112
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7331..7435)
                     /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 gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat ggtgagcaag
     1021 agcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcttggaggg ctccatgaac
     1081 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     1141 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc
     1201 cagatcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacgtccc cgattacatg
     1261 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgcacttcga ggacggcggt
     1321 ctggtgaccg tgactcagga cacctccctg caggacggca cgctgatcta caaggtgaag
     1381 atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac cttgggctgg
     1441 gattatgcca ccgagcgcct gtaccccgaa gacggcgtgc tgaagggcga gcttctgggg
     1501 cgcctgaagc tgaaggacgg cggcctcaac ctggtggagt tcaagaccat ctacatggcc
     1561 aagaagcccg tgcaactgcc cggctactac ttcgtggaca ccaagctggg catcacctcc
     1621 cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg
     1681 gggcatggca ccggcagcag atctatggcg gacgaggaga agctgccgcc cggctgggag
     1741 aagcgcatga gccgcagctc aggccgagtg tactacttca accacatcac taacgccagc
     1801 cagtgggagc ggcccagcgg caacagcagc agtggtggca aaaacgggca gggggagcct
     1861 gccaggggta ccagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt
     1921 agtgctggtg gttccggcag tgctggtggt agtgctggtg gtagtaccag tgctggtggt
     1981 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggttc cggcagtgct
     2041 ggtggtagtg ctggtggtag taccagtgct ggtggtagtg ctggtggtag tgctggtggt
     2101 agtgctggtg gtagtgctgg tggttccggc agtgctggtg gtagtgctgg tggtagtacc
     2161 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt
     2221 tccggaccag atgtccctag agccccagtg gataaagcaa agctgtcatt ccaattcccc
     2281 cgcgcacggg agctcgcctc ctccgaggac aacaacatgg ccaccatcaa agagttcatg
     2341 cgcttcaagg tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag
     2401 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc
     2461 cccctgccct tcgcctggga catcctgtcc ccccagttca tgtacggctc cgaggcgtac
     2521 gtgaggcacc ccgccgacat ccccgattac aagaagctgc ccttccccga gggcttcaag
     2581 tgggagcgcg tgatgaactt cgaagacggc ggtctggtga ccgttaccca ggactcctcc
     2641 ctgcaggacg gcacgctgat ctgcaaggtg aagatgcgcg gcaccaactt cccccccgac
     2701 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct ccaccgagat gctgtacccc
     2761 gaagacggcg tgctgaaggg ccatagctat caggccctga agctgaagga cggcggccac
     2821 tacctggtgg agttcgagac catctacatg gccaagaagc ccgtgcaact gcccggcgat
     2881 tactgtgtgg acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa
     2941 cagtacgggc gctccgaggg ccgccaccgt ctgggcatgg acgagcggta caaggaattc
     3001 taatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     3061 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3121 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3181 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3241 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3301 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3361 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3421 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3481 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3541 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3601 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3661 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3721 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3781 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3841 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3901 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3961 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4021 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4081 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     4141 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4201 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4261 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4321 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4381 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4441 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4501 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4561 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4621 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4681 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4741 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4801 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4861 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4921 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4981 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5041 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5101 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5161 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5221 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5281 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5341 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5401 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5461 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5521 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5581 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5641 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5701 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5761 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5821 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5881 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5941 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6001 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6061 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6121 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6181 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6241 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6301 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6361 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6421 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6481 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6541 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6601 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6661 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6721 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6781 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6841 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6901 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6961 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7021 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7081 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7141 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7201 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7261 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7321 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7381 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7441 ttccccgaaa agtgccacct gacgtc
//
