LOCUS       sequence-399531-        6354 bp    DNA     circular SYN 14-APR-2025
DEFINITION  expresses fluorescent protein-tagged enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  AUTHORS   Wills RC, Doyle CP, Zewe JP, Pacheco J, Hansen SD, Hammond GRV
  TITLE     A novel homeostatic mechanism tunes PI(4,5)P2-dependent signaling at
            the plasma membrane.
  JOURNAL   J Cell Sci. 2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494.
  PUBMED    37534432
REFERENCE   2  (bases 1 to 6354)
  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: "J Cell Sci.
            2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494."
COMMENT     Sequence Label: EGFP-PIP5K1B
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             137..931
                     /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             137..931
                     /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(191..210)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     801..820
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1119..1138)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1163..1210
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1539..2127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2028..2047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2302..2605
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2606..2809
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2760..2780
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2848..2857
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2854..2856,2857..2859,2860..3570)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2854..2856,2857..2859,2860..3570)
                     /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(2899..2920)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3160..3179)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3507..3528
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5383..5504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5420..5439)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5474..5493
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5511..5966)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5648..5669)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5860..5879
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5993..6097
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6099..6354,1..102)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6099..6290
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6122..6142)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(6307..6354,1..88)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
       61 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
      121 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
      181 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
      241 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
      301 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
      361 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
      421 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
      481 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
      541 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
      601 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
      661 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
      721 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
      781 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
      841 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
      901 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
      961 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     1021 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     1081 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     1141 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     1201 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     1261 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     1321 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     1381 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     1441 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1501 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1561 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1621 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1681 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1741 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1801 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1861 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1921 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1981 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2041 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2101 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2161 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2221 aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt cattagttca
     2281 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     2341 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     2401 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     2461 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     2521 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     2581 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     2641 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     2701 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     2761 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
     2821 ccgtcagatc cgctagcgct accggtcgcc accatggtga gcaagggcga ggagctgttc
     2881 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2941 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3001 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     3061 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3121 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3181 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3241 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3301 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     3361 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3421 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     3481 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3541 atcactctcg gcatggacga gctgtacaag tccggactca gatctcgagc tcaagcttcg
     3601 aattctgcag tcgacggtac cgcgatgtct tctgctgctg aaaatggaga ggcagcacct
     3661 ggaaaacaaa atgaagaaaa aacctataaa aagactgcat catctgctat taaaggtgct
     3721 attcagctgg gaataggata cacagtgggt aatctcactt ccaagccaga acgagatgtt
     3781 cttatgcaag acttttatgt ggtggaaagt gtgttcctac ccagcgaagg gagcaatctg
     3841 accccagcac atcactaccc agactttaga tttaagacat acgctccatt agcattccga
     3901 tatttcagag aactttttgg tatcaagcct gatgattact tgtattccat ctgcagtgaa
     3961 cctctaatag aactgtctaa ccctggagcc agtggatcct tgttttttgt gaccagtgat
     4021 gatgaattta tcatcaaaac agttcagcac aaagaagctg agtttcttca gaagctactg
     4081 ccaggctatt acatgaattt aaaccagaat ccaaggactc ttttgccaaa attttacgga
     4141 ctgtattgta tgcaatcagg aggcattaat atcaggattg tggtgatgaa caacgttttg
     4201 ccacgctcca tgagaatgca ctttacatat gacttgaaag gctcaacgta taagcgaaga
     4261 gcatcccgta aagagagaga gaaatccaac cccacattta aggacttaga tttcctgcaa
     4321 gacatgcacg aagggttgta ttttgatacg gaaacataca acgcgcttat gaaaacactt
     4381 cagagagact gccgggtgct agaaagcttc aagatcatgg attatagcct tctgttggga
     4441 attcatttcc tggaccattc cctcaaagag aaagaggagg agaccccaca aaatgtgcct
     4501 gatgctaagc ggactgggat gcagaaggtt ctctactcaa cagccatgga atctatccag
     4561 ggtccaggga aatctggaga tgggataatc acagagaacc cagacacaat gggaggcatt
     4621 ccagctaaaa gccatagggg agaaaaacta cttttattta tgggcattat tgacattctg
     4681 caatcatata ggttaatgaa gaagttagaa cattcctgga aagctcttgt ttatgatggg
     4741 gacactgttt ctgttcatag accaagcttt tatgcagaca gatttcttaa gttcatgaat
     4801 tccagagttt tcaagaaaat tcaagctttg aaggcttcac cgtctaagaa acggtgcaat
     4861 tcaatcgccg ccctaaaggc cacttcacag gagattgtgt cctcaattag ccaggaatgg
     4921 aaggatgaga agcgggattt gctgactgaa ggacaaagtt ttagcagcct tgatgaagaa
     4981 gccctgggat cccgacacag gccagacctg gtccctagca ctccatcact gtttgaagct
     5041 gcttccttgg caaccacaat ttcatcttct tccttatacg tcaatgagca ctatccacac
     5101 gacaggccta cactctattc aaacagcaaa gggttacctt ccagttcaac atttaccttg
     5161 gaagagggga ccatctactt gaccgctgag cccaacactc tggaagtgca ggatgacaat
     5221 gcttctgtgc ttgacgtcta tttataacgc ccgggatcca ccggatctag ataactgatc
     5281 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     5341 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     5401 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5461 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     5521 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5581 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5641 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5701 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5761 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5821 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5881 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5941 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     6001 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6061 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     6121 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     6181 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     6241 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     6301 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggc
//
