LOCUS       sequence-434108-        7065 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Recruitable PIP5K to make PIP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7065)
  AUTHORS   Weckerly CC, Rahn TA, Ehrlich M, Wills RC, Pemberton JG, Airola MV, 
            Hammond GRV
  TITLE     Nir1-LNS2 is a novel phosphatidic acid biosensor that reveals 
            mechanisms of lipid production.
  JOURNAL   bioRxiv [Preprint]. 2024 Feb 28:2024.02.28.582557. doi: 
            10.1101/2024.02.28.582557.
  PUBMED    38464273
REFERENCE   2  (bases 1 to 7065)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Feb 28:2024.02.28.582557. doi: 
            10.1101/2024.02.28.582557."
COMMENT     Sequence Label: pTagBFP-C1-FKBP-PIP5K1C-HD-EE
FEATURES             Location/Qualifiers
     source          1..7065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..363
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        364..567
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     518..538
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      606..615
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             612..1313
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     CDS             1374..1694
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    3852..3973
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3889..3908)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3943..3962
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3980..4435)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4117..4138)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4329..4348
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4462..4566
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4568..4925
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4568..4759
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4591..4611)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4776..4911
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4960..5754
                     /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             4960..5754
                     /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(5014..5033)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5624..5643
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5942..5961)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5986..6033
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6362..6950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6851..6870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
       61 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      121 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      181 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      241 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      301 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      361 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      421 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      481 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      541 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagcgctac
      601 cggtcgccac catggtgagc gagctgatta aggagaacat gcacatgaag ctgtacatgg
      661 agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg
      721 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
      781 acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca
      841 tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
      901 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
      961 tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga
     1021 aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag
     1081 gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca aacgccaaga
     1141 ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg
     1201 actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg
     1261 tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt aattccggac
     1321 tcagatctcg aagcgcggcc gcgggagcag gaggagcagc tcgagcggcg ctgggagtgc
     1381 aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc cagacctgcg
     1441 tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc cgggacagaa
     1501 acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg gaagaagggg
     1561 ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat tatgcctatg
     1621 gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc gatgtggagc
     1681 ttctaaaact ggaaagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg
     1741 gtagtgctgg tggtccgcga gctcaagctt cgaattctgc agtcgacctg caggcgatgg
     1801 agctggaggt accggacgag gcagagagcg ctgaggcagg ggctgtgccc tcggaggcag
     1861 cttgggctgc tgagagcggg gctgcagcag gtttggctca gaagaaggcg gccccaacag
     1921 aggttctgtc catgacggca cagccgggcc ctggccatgg gaagaagttg ggccatcgag
     1981 gtgtggacgc atccggcgaa accacctaca agaagaccac ctcctccacc ctgaagggtg
     2041 ccatccagct gggcatcggc tacaccgtgg gccacctgag ctccaagccc gaacgccgcg
     2101 tgctcatgca ggacttctac gtggtggaga gcatcttctt ccccagcgaa ggcagcaacc
     2161 tcacccccgc ccaccacttc caggacttcc gcttcaagac ctatgcacct gtcgccttcc
     2221 gctacttccg ggagctcttt gggatccggc cagatgatta cttgtactcc ctgtgcaatg
     2281 agccgctgat cgagctgtcc aacccgggcg ccagtggctc cctcttctac gtcaccagcg
     2341 acgacgagtt catcatcaag accgtcatgc acaaggaggc cgagttcctg cagaagctgc
     2401 tccctggcta ctacatgaac ctcaaccaga acccgcggac gctgctgccc aagttctatg
     2461 ggctgtactg cgtgcagtcg gggggcaaga acatccgcgt cgtggtcatg aacaacatcc
     2521 tgccccgcgt ggtcaagatg cacctcaagt tcgacctcaa gggctccacc tacaagcggc
     2581 gcgccagcaa gaaggagaag gagaagagct tccccaccta caaggacctg gacttcatgc
     2641 aggacatgcc cgaggggctc ctgctggacg ccgacacctt cagcgccctg gtcaagacgc
     2701 tgcaggacga ctgcctggtc ctggaaagtt tcaagatcat ggactacagc ctgctgctgg
     2761 gcgtgcacaa catcgaccag cacgagcgcg agcggcaggc gcagggcgcc cagagcacct
     2821 cagatgagaa gcggcctgtg ggccagaagg cgctctactc cacggccatg gagtccatcc
     2881 agggtggcgc agctcgcggg gaggcaatcg aatcggatga cacgatgggc gggatccccg
     2941 ctgtgaacgg ccgcggggag cggctgctgc tgcacattgg catcatcgac atcctgcagt
     3001 cctacaggtt catcaagaaa ctggagcaca cctggaaggc cctcgtccac gatggggaca
     3061 cggtgtccgt ccaccgcccc agcttctatg ccgagcgctt tttcaagttc atgagcaaca
     3121 cggtctttga ggagaactcc tccctgaagt cctcgccctc caagaagggg cgcggcggag
     3181 ccttgctagc tgtgaaacca ctaggaccta ccgctgcatt ctctgcaagt cagatcccta
     3241 gtgaacgaga ggaggctcag tacgacctta gaggggcccg cagttacccc acgctggaag
     3301 acgaaggccg tcctgacctc ctgccttgca cgccaccttc tttcgaagaa gccactacag
     3361 cctccattgc cacgactctg tcatccacat ccctctccat tcctgaacgg tccccttcgg
     3421 aaacgtctga acagccacgg tacaggcggc gcacacagtc atctggacag gatggcaggc
     3481 cgcaggagga gccacccgcg gaagaggatc tgcagcagat tacagtgcag gtggagcctg
     3541 cgtgcagcgt ggagattgtg gtccccaaag aggaggacgc aggggtggag gcttccccgg
     3601 ccggtgcctc tgctgctgtt gaagtagaaa ctgccagcca ggcctcagac gaggagggcg
     3661 cacctgcaag ccaggcatcg gacgaggagg acgcgcccgc caccgacatc tacttttgag
     3721 cgggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     3781 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     3841 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3901 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3961 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     4021 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     4081 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     4141 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     4201 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     4261 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     4321 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     4381 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     4441 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4501 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4561 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     4621 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4681 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4741 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     4801 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4861 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4921 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4981 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     5041 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     5101 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     5161 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     5221 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     5281 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     5341 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     5401 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     5461 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     5521 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     5581 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     5641 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     5701 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     5761 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     5821 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5881 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     5941 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     6001 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     6061 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     6121 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     6181 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     6241 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6301 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6361 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6421 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6481 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6541 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6601 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6661 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6721 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6781 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6841 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6901 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6961 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7021 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcatt
//
