LOCUS       sequence-434080-        4866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Spo20-PABD PA biosensor with a flexible linker to aid in nuclear 
            export.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4866)
  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 4866)
  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: pNES-EGFP-C1-flex-PABD-Spo20
FEATURES             Location/Qualifiers
     source          1..4866
                     /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      595..604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(658..660,661..663,664..1374)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(658..660,661..663,664..1374)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(703..724)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(964..983)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1311..1332
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1654..1775
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1691..1710)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1745..1764
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1782..2237)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1919..1940)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2131..2150
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2264..2368
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2370..2727
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2370..2561
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2393..2413)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2578..2713
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2762..3556
                     /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             2762..3556
                     /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(2816..2835)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3426..3445
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3744..3763)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3788..3835
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4164..4752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4653..4672
                     /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 atggctctgc agaaaaagtt ggaagagctt gagctggatg aggcaccggt cgccaccatg
      661 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      721 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      781 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      841 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      901 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      961 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1021 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1081 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1141 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1201 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1261 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1321 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga
     1381 ggaggatctg gaggatctat ggacaactgt tctgggtcaa gacgccggga tcgccttcac
     1441 gttaaattga aaagtctccg gaataagatt cacaagcagc tgcaccccaa ttgtcgcttc
     1501 gatgacgcga cgaagacaag ctaaggatcc accggatcta gataactgat cataatcagc
     1561 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     1621 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     1681 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     1741 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat
     1801 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1861 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     1921 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1981 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2041 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2101 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2161 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2221 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2281 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2341 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     2401 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2461 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2521 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2581 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     2641 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     2701 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     2761 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     2821 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     2881 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     2941 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3001 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3061 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3121 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3181 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3241 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     3301 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3361 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3421 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3481 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3541 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     3601 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3661 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3721 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     3781 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     3841 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     3901 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     3961 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     4021 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4081 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4141 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4201 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4261 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4321 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4381 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4441 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4501 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4561 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4621 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4681 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4741 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4801 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4861 atgcat
//
