LOCUS       sequence-230370-        7454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative-control mutant for RAB-AKARev biosensor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7454)
  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 7454)
  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-AKARev(T/A)
FEATURES             Location/Qualifiers
     source          1..7454
                     /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"
     promoter        complement(3007..3025)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3008..3025)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3045..3062)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3051..3275
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3321..3749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3408..3427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3618..3639
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3758..3778)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3763..4092
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3943..4078
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4005..4024
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4159..4953
                     /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(4213..4232)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4823..4842
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5127..5248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5164..5183)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5218..5237
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5297..5313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5297..5313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5310..5332)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5321..5337
                     /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(5345..5351,5352..5369,5370..5375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5390..5411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5528..5545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5699..6287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5779..5798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6458..7249,7250..7318))
                     /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     7081..7100
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7319..7423)
                     /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 atctatgcat aagttttctc aagaacagat cggcgaaaac
     1741 attgtgtgca gggtcatttg taccacgggt caaattccca tccgagattt gtcagctgat
     1801 atttcacaag tgcttaagga aaaacgatcc ataaagaaag tttggacatt tggtagaaac
     1861 ccagcctgtg actatcattt aggaaacatt tcaagactgt caaataagca tttccaaata
     1921 ctactaggag aagacggtaa ccttttattg aatgacattt ccactaatgg gacctggtta
     1981 aatgggcaaa aagtcgagaa gaacagcaat cagttactgt cccaaggtga tgaaataacc
     2041 gttggtgtag gcgtggaatc agatatttta tctctggtca ttttcataaa cgacaaattt
     2101 aagcagtgcc tggagcagaa caaagttgat cgcggtacca gtgctggtgg tagtgctggt
     2161 ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggattgag gcgcgcggcc
     2221 ctggttgacg gcggcagcga gctcgcctcc tccgaggaca acaacatggc caccatcaaa
     2281 gagttcatgc gcttcaaggt gcgcatggag ggctccatga acggccacga gttcgagatc
     2341 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     2401 aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcat gtacggctcc
     2461 gaggcgtacg tgaggcaccc cgccgacatc cccgattaca agaagctgcc cttccccgag
     2521 ggcttcaagt gggagcgcgt gatgaacttc gaagacggcg gtctggtgac cgttacccag
     2581 gactcctccc tgcaggacgg cacgctgatc tgcaaggtga agatgcgcgg caccaacttc
     2641 ccccccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagatg
     2701 ctgtaccccg aagacggcgt gctgaagggc catagctatc aggccctgaa gctgaaggac
     2761 ggcggccact acctggtgga gttcgagacc atctacatgg ccaagaagcc cgtgcaactg
     2821 cccggcgatt actgtgtgga caccaagctg gacatcacct cccacaacga ggactacacc
     2881 atcgtggaac agtacgggcg ctccgagggc cgccaccgtc tgggcatgga cgagcggtac
     2941 aagtaagaat tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg
     3001 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc
     3061 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3121 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3181 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3241 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3301 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3361 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3421 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3481 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3541 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3601 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3661 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3721 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3781 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3841 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3901 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3961 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4021 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4081 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     4141 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4201 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4261 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4321 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     4381 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4441 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4501 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4561 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4621 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4681 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4741 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4801 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4861 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4921 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4981 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     5041 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5101 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5161 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5221 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5281 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5341 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5401 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5461 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5521 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5581 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5641 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5701 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5761 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5821 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5881 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5941 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6001 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6061 acaggattag caaagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6121 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6181 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6241 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6301 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6361 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6421 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6481 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6541 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6601 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6661 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6721 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6781 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6841 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6901 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6961 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7021 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7081 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7141 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7201 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7261 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7321 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7381 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7441 tgccacctga cgtc
//
