LOCUS       sequence-413169-        6229 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of y-ATOM biosensor for HRas.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6229)
  AUTHORS   Sekhon H, Ha JH, Presti MF, Procopio SB, Jarvis AR, Mirsky PO, John 
            AM, Loh SN
  TITLE     Adaptable, turn-on maturation (ATOM) fluorescent biosensors for 
            multiplexed detection in cells.
  JOURNAL   Nat Methods. 2023 Nov 9. doi: 10.1038/s41592-023-02065-w.
  PUBMED    37945909
REFERENCE   2  (bases 1 to 6229)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2023 Nov 9. doi: 10.1038/s41592-023-02065-w."
COMMENT     Sequence Label: pCMV-MBP-yATOM_Ras
FEATURES             Location/Qualifiers
     source          1..6229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             642..1742
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1716..1739
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1755..1778
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     1886..1907
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2013..2034)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    3017..3138
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3054..3073)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3108..3127
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3145..3600)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3282..3303)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3494..3513
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3627..3731
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3733..4090
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3733..3924
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3756..3776)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3941..4076
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4125..4919
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4125..4919
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4179..4198)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4789..4808
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5107..5126)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5151..5198
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5527..6115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6016..6035
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgccac catgaaaact gaagaaggta
      661 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
      721 aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga
      781 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
      841 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
      901 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
      961 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc
     1021 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
     1081 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
     1141 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
     1201 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
     1261 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     1321 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     1381 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     1441 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     1501 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     1561 cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga
     1621 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     1681 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     1741 ctaattcgag ctcgctggaa gttctgttcc aggggcccaa ggtaccagga gctgcgacac
     1801 cggtaactgc agacatgctg cccgacaacc actacctgag ctatcagtcc gttctgagca
     1861 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1921 tcactctcgg catggacgag ctgtacaagg gcggtggatc cggtggtatg gtgagcaagg
     1981 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2041 gccacaagtt cagcgtccgc ggcgagggcg agggcgatgc caccaacggc aagctgaccc
     2101 tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     2161 tgggctacgg cctggcctgc ttcagccgct accccgacca catgaagcag cacgacttct
     2221 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctctttc aaggacgacg
     2281 gtacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2341 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2401 acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc atcaaggcta
     2461 acttcaagat ccgccacaac gttgaggacg gtgtggacta cacgattacc gtgtatgcgt
     2521 ggggttggca tggccaggtt tattactata tgggtagccc aatctcgatt aactaccgta
     2581 ccggaggttc aggcagtggt ggtgctagcg gtggtgcgac tggaggcagt ggcggtggat
     2641 cttctgttcc gaccaaactg gaggtggttg ccgcaactcc cacgagtctg ctgatctcat
     2701 gggatgctcc agccgttacc gtcgattatt acgtcatcac ctatggcgaa acaggcggca
     2761 actctccggt acagaagttt gaagtgcctg ggtccaaaag cactgcgacc atttcgggct
     2821 taaaaccggg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2881 gccccgtgct gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga
     2941 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     3001 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3061 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3121 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     3181 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3241 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3301 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3361 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3421 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3481 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3541 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3601 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3661 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3721 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     3781 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3841 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3901 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3961 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4021 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4081 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4141 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4201 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4261 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     4321 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4381 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4441 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4501 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4561 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4621 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     4681 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4741 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4801 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4861 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4921 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4981 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5041 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     5101 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     5161 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     5221 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     5281 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     5341 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     5401 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5461 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5521 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5581 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5641 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5701 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5761 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5821 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5881 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5941 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6001 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6061 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6121 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6181 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
