LOCUS       sequence-434082-        5614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High affinity PA biosensor with green fluorophore (also known as 
            NeonGreen-PILS-Nir1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5614)
  AUTHORS   Weckerly CC, Rahn TA, Ehrlich M, Wills RC, Pemberton JG, Airola MV, 
            Hammond GRV
  TITLE     PILS-Nir1 is a sensitive phosphatidic acid biosensor that reveals 
            mechanisms of lipid production.
  JOURNAL   J Cell Biol. 2025 Nov 3;224(11):e202405174. doi: 
            10.1083/jcb.202405174. Epub 2025 Sep 9.
  PUBMED    40923975
REFERENCE   2  (bases 1 to 5614)
  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: "J Cell 
            Biol."; date: "2025-11-3"; volume: "224(11):e202405174. doi"; pages:
            " 10.1083/jcb.202405174. Epub 2025 Sep 9"
COMMENT     Sequence Label: pNeonGreen-C1-Nir1-LNS2
FEATURES             Location/Qualifiers
     source          1..5614
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1320
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      868..877
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    2213..2278
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2402..2523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2439..2458)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2493..2512
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2530..2985)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2667..2688)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2879..2898
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3012..3116
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3118..3475
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3118..3309
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3141..3161)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3326..3461
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3510..4304
                     /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             3510..4304
                     /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(3564..3583)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4174..4193
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4492..4511)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4536..4583
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4912..5500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5401..5420
                     /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 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggcctctct cccagcgaca
      661 catgagttac acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc
      721 accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc
      781 cagttctccc cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc
      841 taccctgacg ggatgtcgcc tttccaggcc gccatggtag atggctccgg ataccaagtc
      901 catcgcacaa tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac
      961 gagggaagcc acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt
     1021 cctgtgatga ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc
     1081 aacgacaaaa ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc
     1141 taccggagca ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg
     1201 aagaaccagc cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc
     1261 aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag
     1321 ggaggctcag gaggcatgcc tgctaacccg cgtgaaaaat ggctgcgtaa acgtacccag
     1381 gttaaactgc gtaacgttac cgctaaccac cgtgcgaacg atgttatcgc ggcggaagat
     1441 ggtccgcagg ttctggttgg tcgtttcatg tacggtccgc tggacatggt tgctctgacc
     1501 ggtgaaaaag tggatatcct ggttatggca gaaccgtcta gcggtcgctg ggttcacctg
     1561 gacactgaaa tcaccaactc ctctggtcgt atcacctaca acgttccgcg tccgcgccgc
     1621 ctgggtgttg gtgtttaccc ggttaaaatg gttgttcgtg gtgaccagac ctgcgcgatg
     1681 tcttacctga ccgttctgcc gcgcggtatg gaatgtgttg tgttctctat cgacggttct
     1741 ttcgctgcga gcgtttctat catgggctct gacccgaaag ttcgtccggg tgcggttgac
     1801 gttgttcgtc actggcagga cctgggttac atgatcctgt acatcaccgg ccgtccggac
     1861 atgcagaaac agcgtgttgt ttcttggctg tctcagcaca acttcccgca gggtatgatc
     1921 ttcttctctg atggtctggt tcacgacccg ctgcgtcaga aagcgatctt cctgcgtaac
     1981 ctgatgcagg aatgcttcat caaaatctct gcggcgtacg gttctaccaa agacatctct
     2041 gtttactccg ttctgggtct gccggcttct cagatcttca tcgtgggtcg tccgaccaaa
     2101 aaataccaga cccagtgtca gttcctgtct gaaggttacg cggcgcacct ggctgttctg
     2161 gaagcttctc accgttctcg tccgaaaaag aactaagcgg ccgctcgagt cttccggact
     2221 cagatctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc cgggatccac
     2281 cggatctaga taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt
     2341 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2401 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2461 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2521 ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2581 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2641 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     2701 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     2761 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2821 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2881 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2941 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     3001 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3061 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     3121 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3181 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3241 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3301 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3361 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3421 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     3481 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3541 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3601 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3661 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     3721 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3781 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3841 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3901 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3961 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4021 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4081 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4141 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4201 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4261 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4321 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4381 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4441 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     4501 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     4561 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     4621 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     4681 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     4741 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     4801 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4861 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4921 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4981 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5041 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5101 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5161 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5221 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5281 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5341 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5401 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5461 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5521 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5581 tgattctgtg gataaccgta ttaccgccat gcat
//
