LOCUS       sequence-434103-        4733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal amphipathic helix of Nir1-LNS2 domain; loses ability to 
            bind PA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4733)
  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 4733)
  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-N1-Nir1-613-630
FEATURES             Location/Qualifiers
     source          1..4733
                     /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             688..1398
                     /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      943..952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1521..1642
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1558..1577)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1612..1631
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1649..2104)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1786..1807)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1998..2017
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2131..2235
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2237..2594
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2237..2428
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2260..2280)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2445..2580
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2629..3423
                     /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             2629..3423
                     /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(2683..2702)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3293..3312
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3611..3630)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3655..3702
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4031..4619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4520..4539
                     /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 ccaccatgcc tgctaacccg cgtgaaaaat ggctgcgtaa acgtacccag
      661 gttaaactgc gtggaggctc aggaggcatg gtgagcaagg gcgaggagga taacatggcc
      721 tctctcccag cgacacatga gttacacatc tttggctcca tcaacggtgt ggactttgac
      781 atggtgggtc agggcaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc
      841 accaagggtg acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc
      901 catcagtacc tgccctaccc tgacgggatg tcgcctttcc aggccgccat ggtagatggc
      961 tccggatacc aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac
     1021 taccgctaca cctacgaggg aagccacatc aaaggagagg cccaggtgaa ggggactggt
     1081 ttccctgctg acggtcctgt gatgaccaac tcgctgaccg ctgcggactg gtgcaggtcg
     1141 aagaagactt accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact
     1201 ggaaatggca agcgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg
     1261 gcggctaact atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac
     1321 tccaagaccg agctcaactt caaggagtgg caaaaggcct ttaccgatgt gatgggcatg
     1381 gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg
     1441 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     1501 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     1561 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     1621 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc
     1681 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1741 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1801 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1861 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1921 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     1981 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     2041 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     2101 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2161 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2221 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     2281 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2341 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2401 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2461 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2521 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2581 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     2641 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     2701 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     2761 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     2821 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     2881 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     2941 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3001 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3061 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3121 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     3181 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3241 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3301 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3361 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3421 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3481 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3541 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
     3601 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
     3661 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt
     3721 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg
     3781 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca
     3841 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt
     3901 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3961 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4021 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4081 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4141 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4201 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4261 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4321 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4381 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4441 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4501 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4561 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4621 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4681 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
