LOCUS       sequence-382243-        4836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Monitoring the production of phosphatidic acid with RFP-PASS 
            (phosphatidic acid biosensor with superior sensitivity).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4836)
  AUTHORS   Zhang F, Wang Z, Lu M, Yonekubo Y, Liang X, Zhang Y, Wu P, Zhou Y, 
            Grinstein S, Hancock JF, Du G
  TITLE     Temporal production of the signaling lipid phosphatidic acid by 
            phospholipase D2 determines the output of extracellular 
            signal-regulated kinase signaling in cancer cells.
  JOURNAL   Mol Cell Biol. 2014 Jan;34(1):84-95. doi: 10.1128/MCB.00987-13. Epub
            2013 Oct 28.
  PUBMED    24164897
REFERENCE   2  (bases 1 to 4836)
  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: 
            "10.1128/MCB.00987-13"; journalName: "Mol Cell Biol"; date: 
            "2014-01"; volume: "34"; issue: "1"; pages: "84-95"
COMMENT     Sequence Label: RFP-PASS
FEATURES             Location/Qualifiers
     source          1..4836
                     /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             604..621
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             625..1296
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             625..1296
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(752..770)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1020..1039
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1214..1237
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1624..1745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1661..1680)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1715..1734
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1752..2207)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1889..1910)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2101..2120
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2234..2338
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2340..2697
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2340..2531
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2363..2383)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2548..2683
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2732..3526
                     /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             2732..3526
                     /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(2786..2805)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3396..3415
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3714..3733)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3758..3805
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4134..4722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4623..4642
                     /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 gctagccacc
      601 atgcatcacc atcaccatca cggggcctcc tccgaggacg tcatcaagga gttcatgcgc
      661 ttcaaggtgc gcatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
      721 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc
      781 ctgcccttcg cctgggacat cctgtcccct cagttccagt acggctccaa ggcctacgtg
      841 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
      901 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
      961 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1021 cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagcggat gtaccccgag
     1081 gacggcgccc tgaagggcga gatcaagatg aggctgaagc tgaaggacgg cggccactac
     1141 gacgccgagg tcaagaccac ctacatggcc aagaagcccg tgcagctgcc cggcgcctac
     1201 aagaccgaca tcaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag
     1261 tacgagcgcg ccgagggccg ccactccacc ggcgccagat ctcgagcgaa cagcaatgaa
     1321 ttagccttga aattagcagg tcttgatatc aacaagacag aatctagaat ggacaattgt
     1381 tcaggaagca gaagacgtga taggctacat gtgaagctta aatccttgag gaataaaatc
     1441 cacaaacaac ttcacccaaa ctgtcggttc gatgacgcca ctaagactag ttaaggatcc
     1501 accggatcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     1561 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     1621 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1681 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     1741 tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1801 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     1861 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     1921 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     1981 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2041 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2101 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2161 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     2221 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2281 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     2341 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2401 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2461 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2521 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2581 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2641 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     2701 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2761 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     2821 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     2881 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     2941 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3001 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3061 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3121 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3181 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3241 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3301 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3361 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     3421 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     3481 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3541 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3601 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3661 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     3721 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     3781 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     3841 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     3901 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     3961 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     4021 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4081 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4141 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4201 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4261 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4321 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4381 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4441 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4501 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4561 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4621 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4681 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4741 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4801 cctgattctg tggataaccg tattaccgcc atgcat
//
