LOCUS       sequence-360278-        6351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminally tagged Shigella flexneri IpgD (catalytically inactive) 
            for mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6351)
  AUTHORS   Walpole GFW, Pacheco J, Chauhan N, Clark J, Anderson KE, Abbas YM, 
            Brabant-Kirwan D, Montano-Rendon F, Liu Z, Zhu H, Brumell JH, 
            Deiters A, Stephens LR, Hawkins PT, Hammond GRV, Grinstein S, Fairn 
            GD
  TITLE     Kinase-independent synthesis of 3-phosphorylated phosphoinositides 
            by a phosphotransferase.
  JOURNAL   Nat Cell Biol. 2022 Apr 28. pii: 10.1038/s41556-022-00895-y. doi: 
            10.1038/s41556-022-00895-y.
  PUBMED    35484249
REFERENCE   2  (bases 1 to 6351)
  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: "Nat Cell 
            Biol. 2022 Apr 28. pii: 10.1038/s41556-022-00895-y. doi: 
            10.1038/s41556-022-00895-y."
COMMENT     Sequence Label: EGFP-IpgD(C439S)
FEATURES             Location/Qualifiers
     source          1..6351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        76..379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        380..583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     534..554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      622..631
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(628..630,631..633,634..1344)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(628..630,631..633,634..1344)
                     /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(673..694)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(934..953)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1281..1302
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3154..3275
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3191..3210)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3245..3264
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3282..3737)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3419..3440)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3631..3650
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3764..3868
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3870..4227
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3870..4061
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3893..3913)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4078..4213
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4262..5056
                     /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             4262..5056
                     /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(4316..4335)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4926..4945
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5244..5263)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5288..5335
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5664..6252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6153..6172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc
       61 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      121 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      181 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      241 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      301 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      361 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      421 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      481 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      541 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
      601 gatccgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
      661 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
      721 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
      781 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
      841 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
      901 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
      961 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1021 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1081 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1141 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1201 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1261 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1321 ctcggcatgg acgagctgta caagtccgga ctcagatctc gagctcaagc ttcgaattct
     1381 gcagtcgacg gtggtagtgc ttccggaatg cacataacta atttgggatt gcatcaggtt
     1441 tcatttcaaa gcggagattc ctataaaggc gcagaagaaa ccggaaagca caaaggtgta
     1501 agcgtgattt catatcaaag agttaaaaat ggagaaagaa ataaggggat cgaagcgctt
     1561 aatcgattat atttacagaa ccaaacaagc ttaacaggaa aaagcctttt gtttgcgcgt
     1621 gacaaagctg aagttttttg cgaggcaata aagttggcag gtggtgatac gtcaaaaatt
     1681 aaagccatga tggaacgatt agatacctat aaacttggtg aagttaataa aagacatatt
     1741 aatgagctta ataaagtaat aagtgaagag ataagagcac agctaggcat taaaaataaa
     1801 aaggaattgc aaacgaaaat aaaacaaata tttactgatt atttaaataa taagaattgg
     1861 ggaccggtca acaagaatat tagtcatcat gggaaaaatt atagttttca attaactcct
     1921 gcctctcata tgaagatagg gaataaaaat atatttgtca aagagtataa tggaaaagga
     1981 atttgttgtg cttctacgag agagcgtgat catatcgcga atatgtggct ttcaaaagtg
     2041 gtggatgacg aggggaagga aattttttcg ggtatcagac atggagttat atcagcctat
     2101 ggattgaaaa aaaactcatc tgaaagagct gttgctgctc gtaataaagc tgaggagtta
     2161 gtaagcgcag cattatatag cagaccagag ttattatcac aggctttgtc tggtaaaaca
     2221 gtagatttaa agattgtttc gacttctctt ctgacgccga ccagtttaac cgggggagag
     2281 gagagtatgt taaaagatca ggtgagcgca ttaaaagggt taaactctaa aagggggggg
     2341 ccaacgaaat tattaattcg gaatagtgat ggtcttctga aagaagtaag cgttaatcta
     2401 aaagtggtga catttaactt tggtgtaaat gaattagcgc taaaaatggg cttaggctgg
     2461 aggaatgttg acaagcttaa tgatgaatca atatgttctt tgttggggga taatttcctc
     2521 aaaaatggtg tgattggagg ctgggctgca gaagcaatag aaaaaaatcc accatgtaag
     2581 aatgatgtga tatatctggc taaccagata aaagagattg taaacaataa gttacaaaaa
     2641 aatgataatg gagagccgta taaattgtca caaagagtga ctctattggc ttatactata
     2701 ggtgctgtac cttgctggaa ttcgaagagt gggaaggaca gaacaggtat gcaggatgct
     2761 gaaattaaaa gagaaataat aagaaaacat gaaacaggtc agttttctca attaaatagt
     2821 aaattatcct cagaagagaa aagattattt tctactattc taatgaatag tggtaatatg
     2881 gaaatccaag agatgaatac tggtgtgccc ggaaataaag tcatgaaaaa attgccgcta
     2941 tcttcgttag agctatctta ttctgaaaga ataggggacc caaaaatatg gaatatggtg
     3001 aaagggtatt cgtcatttgt ataaggatcc accggatcta gataactgat cataatcagc
     3061 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     3121 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     3181 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3241 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat
     3301 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     3361 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     3421 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     3481 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     3541 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     3601 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     3661 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3721 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3781 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3841 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     3901 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3961 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     4021 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4081 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4141 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     4201 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     4261 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4321 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4381 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4441 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4501 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4561 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4621 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4681 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4741 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     4801 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4861 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4921 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4981 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5041 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5101 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5161 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5221 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     5281 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     5341 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     5401 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     5461 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     5521 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5581 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5641 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5701 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5761 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5821 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5881 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5941 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6001 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6061 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6121 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6181 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6241 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6301 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg t
//
