LOCUS       sequence-492975-        5614 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  PILS-Nir1 K820E non-PA binding negative control.
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 Nov 18, 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-K820E
FEATURES             Location/Qualifiers
     source          1..5614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        59..262
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     213..233
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      301..310
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             307..1014
                     /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      562..571
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1907..1972
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2096..2217
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2133..2152)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2187..2206
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2224..2679)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2361..2382)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2573..2592
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2706..2810
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2812..3169
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2812..3003
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2835..2855)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3020..3155
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3204..3998
                     /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             3204..3998
                     /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(3258..3277)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3868..3887
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4186..4205)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4230..4277
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4606..5194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5095..5114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        join(5369..5614,1..58)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
       61 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      121 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      181 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      241 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc
      301 gccaccatgg tgagcaaggg cgaggaggat aacatggcct ctctcccagc gacacatgag
      361 ttacacatct ttggctccat caacggtgtg gactttgaca tggtgggtca gggcaccggc
      421 aatccaaatg atggttatga ggagttaaac ctgaagtcca ccaagggtga cctccagttc
      481 tccccctgga ttctggtccc tcatatcggg tatggcttcc atcagtacct gccctaccct
      541 gacgggatgt cgcctttcca ggccgccatg gtagatggct ccggatacca agtccatcgc
      601 acaatgcagt ttgaagatgg tgcctccctt actgttaact accgctacac ctacgaggga
      661 agccacatca aaggagaggc ccaggtgaag gggactggtt tccctgctga cggtcctgtg
      721 atgaccaact cgctgaccgc tgcggactgg tgcaggtcga agaagactta ccccaacgac
      781 aaaaccatca tcagtacctt taagtggagt tacaccactg gaaatggcaa gcgctaccgg
      841 agcactgcgc ggaccaccta cacctttgcc aagccaatgg cggctaacta tctgaagaac
      901 cagccgatgt acgtgttccg taagacggag ctcaagcact ccaagaccga gctcaacttc
      961 aaggagtggc aaaaggcctt taccgatgtg atgggcatgg acgagctgta caagggaggc
     1021 tcaggaggca tgcctgctaa cccgcgtgaa aaatggctgc gtaaacgtac ccaggttaaa
     1081 ctgcgtaacg ttaccgctaa ccaccgtgcg aacgatgtta tcgcggcgga agatggtccg
     1141 caggttctgg ttggtcgttt catgtacggt ccgctggaca tggttgctct gaccggtgaa
     1201 aaagtggata tcctggttat ggcagaaccg tctagcggtc gctgggttca cctggacact
     1261 gaaatcacca actcctctgg tcgtatcacc tacaacgttc cgcgtccgcg ccgcctgggt
     1321 gttggtgttt acccggttaa aatggttgtt cgtggtgacc agacctgcgc gatgtcttac
     1381 ctgaccgttc tgccgcgcgg tatggaatgt gttgtgttct ctatcgacgg ttctttcgct
     1441 gcgagcgttt ctatcatggg ctctgacccg aaagttcgtc cgggtgcggt tgacgttgtt
     1501 cgtcactggc aggacctggg ttacatgatc ctgtacatca ccggccgtcc ggacatgcag
     1561 aaacagcgtg ttgtttcttg gctgtctcag cacaacttcc cgcagggtat gatcttcttc
     1621 tctgatggtc tggttcacga cccgctgcgt caggaagcga tcttcctgcg taacctgatg
     1681 caggaatgct tcatcaaaat ctctgcggcg tacggttcta ccaaagacat ctctgtttac
     1741 tccgttctgg gtctgccggc ttctcagatc ttcatcgtgg gtcgtccgac caaaaaatac
     1801 cagacccagt gtcagttcct gtctgaaggt tacgcggcgc acctggctgt tctggaagct
     1861 tctcaccgtt ctcgtccgaa aaagaactaa gcggccgctc gagtcttccg gactcagatc
     1921 tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggatc
     1981 tagataactg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     2041 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     2101 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     2161 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg
     2221 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2281 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2341 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2401 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2461 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2521 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2581 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2641 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     2701 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2761 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
     2821 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     2881 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     2941 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3001 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3061 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     3121 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     3181 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3241 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3301 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3361 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     3421 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3481 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3541 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3601 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3661 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3721 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3781 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3841 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3901 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3961 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4021 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4081 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4141 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     4201 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     4261 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     4321 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     4381 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     4441 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4501 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4561 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4621 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4681 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4741 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4801 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4861 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4921 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4981 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5041 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5101 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5161 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5221 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5281 tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat
     5341 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     5401 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     5461 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     5521 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     5581 aatggcccgc ctggcattat gcccagtaca tgac
//
