LOCUS       sequence-363417-        7931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lysosome-targeted ExRai-AktAR2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7931)
  AUTHORS   Chen M, Sun T, Zhong Y, Zhou X, Zhang J
  TITLE     A Highly Sensitive Fluorescent Akt Biosensor Reveals 
            Lysosome-Selective Regulation of Lipid Second Messengers and Kinase 
            Activity.
  JOURNAL   ACS Cent Sci. 2021 Dec 22;7(12):2009-2020. doi: 
            10.1021/acscentsci.1c00919. Epub 2021 Dec 3.
  PUBMED    34963894
REFERENCE   2  (bases 1 to 7931)
  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.1c00919"; journalName: "ACS Cent Sci"; date: 
            "2021-12-22- 22"; volume: "7"; issue: "12"; pages: "2009-2020"
COMMENT     Sequence Label: pcDNA3-Lyso-ExRai-AktAR2
FEATURES             Location/Qualifiers
     source          1..7931
                     /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"
     regulatory      905..914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(907..990,991..2157)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFAYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTPHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     primer_bind     2472..2493
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2599..2620)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2860..2879)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(3484..3502)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3485..3502)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3522..3539)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3528..3752
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3798..4226
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3885..3904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4095..4116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4235..4255)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4240..4569
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4420..4555
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4482..4501
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4636..5430
                     /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(4690..4709)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5300..5319
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5604..5725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5641..5660)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5695..5714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5774..5790)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5774..5790)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5787..5809)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5798..5814
                     /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(5822..5828,5829..5846,5847..5852))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5867..5888
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6005..6022)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6176..6764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6256..6275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6935..7726,7727..7795))
                     /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     7558..7577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7796..7900)
                     /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 cggcgcccgg cagcgcccgg cgacccctgc tgctgctact gctgttgctg
      961 ctgctcggcc tcatgcattg tgcgtcagca gcaatgttta tggtgaaaaa tggcaacggg
     1021 accgcgtgca taatggccaa cttctctgct gccttctcag tgaactacga caccaagagt
     1081 ggccctaaga acatgacctt tgacctgcca tcagatgcca cagtggtgct caaccgcagc
     1141 tcctgtggaa aagagaacac ttctgacccc agtctcgtga ttgcttttgg aagaggacat
     1201 acactcactc tcaatttcac gagaaatgca acacgttaca gcgtccagct catgagtttt
     1261 gcttataact tgtcagacac acaccttttc cccaatgcga gctccaaaga aatcaagact
     1321 gtggaatcta taactgacat cagggcagat atagataaaa aatacagatg tgttagtggc
     1381 acccaggtcc acatgaacaa cgtgaccgta acgccccatg atgccaccat ccaggcgtac
     1441 ctttccaaca gcagcttcag caggggagag acacgctgtg aacaagacag gccttcccca
     1501 accacagcgc cccctgcgcc acccagcccc tcgccctcac ccgtgcccaa gagcccctct
     1561 gtggacaagt acaacgtgag cggcaccaac gggacctgcc tgctggccag catggggctg
     1621 cagctgaacc tcacctatga gaggaaggac aacacgacgg tgacaaggct tctcaacatc
     1681 aaccccaaca agacctcggc cagcgggagc tgcggcgccc acctggtgac tctggagctg
     1741 cacagcgagg gcaccaccgt cctgctcttc cagttcggga tgaatgcaag ttctagccgg
     1801 tttttcctac aaggaatcca gttgaataca attcttcctg acgccagaga ccctgccttt
     1861 aaagctgcca acggctccct gcgagcgctg caggccacag tcggcaattc ctacaagtgc
     1921 aacgcggagg agcacgtccg tgtcacgaag gcgttttcag tcaatatatt caaagtgtgg
     1981 gtccaggctt tcaaggtgga aggtggccag tttggctctg tggaggagtg tctgctggac
     2041 gagaacagca tgctgatccc catcgctgtg ggtggtgccc tggcggggct ggtcctcatc
     2101 gtcctcatcg cctacctcgt cggcaggaag aggagtcacg caggctacca gactatcggt
     2161 ggtggcggtg gtgcggatcc cgtcgaccct cgtccgcgct cgtgcacctg gccggacccc
     2221 aggccggagt ttggaggtac cggcggcagc gagctcagct tgcagaacgt ctatatcaag
     2281 gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac
     2341 ggcggcgtgc agctcgccta ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2401 ctgctgcccg acaaccacta cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag
     2461 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2521 gacgagctgt acaagggcgg taccggaggg agcatggtga gcaagggcga ggagctgttc
     2581 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2641 gtgtccggcg agggtgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     2701 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     2761 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2821 cccgaaggct acatccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2881 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2941 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt gctgcataag
     3001 ttttctcaag aacagatcgg cgaaaacatt gtgtgcaggg tcatttgtac cacgggtcaa
     3061 attcccatcc gagatttgtc agctgatatt tcacaagtgc ttaaggaaaa acgatccata
     3121 aagaaagttt ggacatttgg tagaaaccca gcctgtgact atcatttagg aaacatttca
     3181 agactgtcaa ataagcattt ccaaatacta ctaggagaag acggtaacct tttattgaat
     3241 gacatttcca ctaatgggac ctggttaaat gggcaaaaag tcgagaagaa cagcaatcag
     3301 ttactgtctc aaggtgatga aataaccgtt ggtgtaggcg tggaatcaga tattttatct
     3361 ctggtcattt tcataaacga caaatttaag cagtgcctcg agcagaacaa agttgatcgc
     3421 taagaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc
     3481 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     3541 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3601 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3661 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3721 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3781 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3841 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4021 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4081 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4141 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4201 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     4261 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4321 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4381 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4441 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4501 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4561 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     4621 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4681 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4741 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4801 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4861 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4921 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4981 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     5041 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     5101 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     5161 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     5221 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     5281 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     5341 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     5401 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     5461 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5521 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5581 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5641 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5701 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5761 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5821 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5881 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5941 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6001 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6061 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6121 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6181 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6241 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6301 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6361 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6421 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6481 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6541 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6601 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6661 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6721 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6781 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6841 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6901 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6961 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7021 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7081 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7141 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7201 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7261 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7321 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7381 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7441 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7501 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7561 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7621 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7681 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7741 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7801 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7861 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7921 cacctgacgt c
//
