LOCUS       sequence-434090-        5851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  high affinity PA biosensor with far-red fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5851)
  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 5851)
  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: pmiRFP670-C1-Nir1-LNS2
FEATURES             Location/Qualifiers
     source          1..5851
                     /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..1557
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASTNAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     misc_feature    2450..2515
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2639..2760
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2676..2695)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2730..2749
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2767..3222)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2904..2925)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3116..3135
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3249..3353
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3355..3712
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3355..3546
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3378..3398)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3563..3698
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3747..4541
                     /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             3747..4541
                     /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(3801..3820)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4411..4430
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4729..4748)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4773..4820
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5149..5737
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5638..5657
                     /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 ccatggtagc aggtcatgcc tctggcagcc ccgcattcgg gaccgcctct
      661 cattcgaatt gcgaacatga agagatccac ctcgccggct cgatccagcc gcatggcgcg
      721 cttctggtcg tcagcgaaca tgatcatcgc gtcatccagg ccagcaccaa cgccgcggaa
      781 tttctgaatc tcggaagcgt actcggcgtt ccgctcgccg agatcgacgg cgatctgttg
      841 atcaagatcc tgccgcatct cgatcccacc gccgaaggca tgccggtcgc ggtgcgctgc
      901 cggatcggca atccctctac ggagtactgc ggtctgatgc atcggcctcc ggaaggcggg
      961 ctgatcatcg aactcgaacg tgccggcccg tcgatcgatc tgtcaggcac gctggcgccg
     1021 gcgctggagc ggatccgcac ggcgggttca ctgcgcgcgc tgtgcgatga caccgtgctg
     1081 ctgtttcagc agtgcaccgg ctacgaccgg gtgatggtgt atcgtttcga tgagcaaggc
     1141 cacggcctgg tattctccga gtgccatgtg cctgggctcg aatcctattt cggcaaccgc
     1201 tatccgtcgt cgactgtccc gcagatggcg cggcagctgt acgtgcggca gcgcgtccgc
     1261 gtgctggtcg acgtcaccta tcagccggtg ccgctggagc cgcggctgtc gccgctgacc
     1321 gggcgcgatc tcgacatgtc gggctgcttc ctgcgctcga tgtcgccgtg ccatctgcag
     1381 ttcctgaagg acatgggcgt gcgcgccacc ctggcggtgt cgctggtggt cggcggcaag
     1441 ctgtggggcc tggttgtctg tcaccattat ctgccgcgct tcatccgttt cgagctgcgg
     1501 gcgatctgca aacggctcgc cgaaaggatc gcgacgcgga tcaccgcgct tgagagcgga
     1561 ggctcaggag gcatgcctgc taacccgcgt gaaaaatggc tgcgtaaacg tacccaggtt
     1621 aaactgcgta acgttaccgc taaccaccgt gcgaacgatg ttatcgcggc ggaagatggt
     1681 ccgcaggttc tggttggtcg tttcatgtac ggtccgctgg acatggttgc tctgaccggt
     1741 gaaaaagtgg atatcctggt tatggcagaa ccgtctagcg gtcgctgggt tcacctggac
     1801 actgaaatca ccaactcctc tggtcgtatc acctacaacg ttccgcgtcc gcgccgcctg
     1861 ggtgttggtg tttacccggt taaaatggtt gttcgtggtg accagacctg cgcgatgtct
     1921 tacctgaccg ttctgccgcg cggtatggaa tgtgttgtgt tctctatcga cggttctttc
     1981 gctgcgagcg tttctatcat gggctctgac ccgaaagttc gtccgggtgc ggttgacgtt
     2041 gttcgtcact ggcaggacct gggttacatg atcctgtaca tcaccggccg tccggacatg
     2101 cagaaacagc gtgttgtttc ttggctgtct cagcacaact tcccgcaggg tatgatcttc
     2161 ttctctgatg gtctggttca cgacccgctg cgtcagaaag cgatcttcct gcgtaacctg
     2221 atgcaggaat gcttcatcaa aatctctgcg gcgtacggtt ctaccaaaga catctctgtt
     2281 tactccgttc tgggtctgcc ggcttctcag atcttcatcg tgggtcgtcc gaccaaaaaa
     2341 taccagaccc agtgtcagtt cctgtctgaa ggttacgcgg cgcacctggc tgttctggaa
     2401 gcttctcacc gttctcgtcc gaaaaagaac taagcggccg ctcgagtctt ccggactcag
     2461 atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg gatccaccgg
     2521 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     2581 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     2641 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2701 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2761 aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2821 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2881 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2941 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     3001 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     3061 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     3121 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     3181 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     3241 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3301 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     3361 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3421 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3481 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3541 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3601 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     3661 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     3721 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3781 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3841 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3901 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     3961 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4021 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4081 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4141 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4201 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4261 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4321 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4381 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4441 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4501 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     4561 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4621 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4681 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     4741 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     4801 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     4861 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     4921 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     4981 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5041 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5101 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5161 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5221 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5281 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5341 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5401 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5461 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5521 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5581 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5641 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5701 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5761 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5821 ttctgtggat aaccgtatta ccgccatgca t
//
