LOCUS       sequence-360255-        6351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminally tagged Shigella flexneri IpgD 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
FEATURES             Location/Qualifiers
     source          1..6351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        70..373
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        374..577
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     528..548
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      616..625
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(622..624,625..627,628..1338)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(622..624,625..627,628..1338)
                     /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(667..688)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(928..947)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1275..1296
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3148..3269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3185..3204)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3239..3258
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3276..3731)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3413..3434)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3625..3644
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3758..3862
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3864..4221
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3864..4055
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3887..3907)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4072..4207
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4256..5050
                     /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             4256..5050
                     /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(4310..4329)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4920..4939
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5238..5257)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5282..5329
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5658..6246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6147..6166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
       61 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      121 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      181 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      241 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      301 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      361 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      421 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      481 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      541 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg
      601 ctagcgctac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
      661 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      721 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      781 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
      841 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      901 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      961 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1021 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1081 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1141 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1201 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     1261 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1321 atggacgagc tgtacaagtc cggactcaga tctcgagctc aagcttcgaa ttctgcagtc
     1381 gacggtggta gtgcttccgg aatgcacata actaatttgg gattgcatca ggtttcattt
     1441 caaagcggag attcctataa aggcgcagaa gaaaccggaa agcacaaagg tgtaagcgtg
     1501 atttcatatc aaagagttaa aaatggagaa agaaataagg ggatcgaagc gcttaatcga
     1561 ttatatttac agaaccaaac aagcttaaca ggaaaaagcc ttttgtttgc gcgtgacaaa
     1621 gctgaagttt tttgcgaggc aataaagttg gcaggtggtg atacgtcaaa aattaaagcc
     1681 atgatggaac gattagatac ctataaactt ggtgaagtta ataaaagaca tattaatgag
     1741 cttaataaag taataagtga agagataaga gcacagctag gcattaaaaa taaaaaggaa
     1801 ttgcaaacga aaataaaaca aatatttact gattatttaa ataataagaa ttggggaccg
     1861 gtcaacaaga atattagtca tcatgggaaa aattatagtt ttcaattaac tcctgcctct
     1921 catatgaaga tagggaataa aaatatattt gtcaaagagt ataatggaaa aggaatttgt
     1981 tgtgcttcta cgagagagcg tgatcatatc gcgaatatgt ggctttcaaa agtggtggat
     2041 gacgagggga aggaaatttt ttcgggtatc agacatggag ttatatcagc ctatggattg
     2101 aaaaaaaact catctgaaag agctgttgct gctcgtaata aagctgagga gttagtaagc
     2161 gcagcattat atagcagacc agagttatta tcacaggctt tgtctggtaa aacagtagat
     2221 ttaaagattg tttcgacttc tcttctgacg ccgaccagtt taaccggggg agaggagagt
     2281 atgttaaaag atcaggtgag cgcattaaaa gggttaaact ctaaaagggg ggggccaacg
     2341 aaattattaa ttcggaatag tgatggtctt ctgaaagaag taagcgttaa tctaaaagtg
     2401 gtgacattta actttggtgt aaatgaatta gcgctaaaaa tgggcttagg ctggaggaat
     2461 gttgacaagc ttaatgatga atcaatatgt tctttgttgg gggataattt cctcaaaaat
     2521 ggtgtgattg gaggctgggc tgcagaagca atagaaaaaa atccaccatg taagaatgat
     2581 gtgatatatc tggctaacca gataaaagag attgtaaaca ataagttaca aaaaaatgat
     2641 aatggagagc cgtataaatt gtcacaaaga gtgactctat tggcttatac tataggtgct
     2701 gtaccttgct ggaattgtaa gagtgggaag gacagaacag gtatgcagga tgctgaaatt
     2761 aaaagagaaa taataagaaa acatgaaaca ggtcagtttt ctcaattaaa tagtaaatta
     2821 tcctcagaag agaaaagatt attttctact attctaatga atagtggtaa tatggaaatc
     2881 caagagatga atactggtgt gcccggaaat aaagtcatga aaaaattgcc gctatcttcg
     2941 ttagagctat cttattctga aagaataggg gacccaaaaa tatggaatat ggtgaaaggg
     3001 tattcgtcat ttgtataagg atccaccgga tctagataac tgatcataat cagccatacc
     3061 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     3121 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     3181 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3241 ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt
     3301 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3361 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3421 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3481 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3541 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3601 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3661 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3721 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3781 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3841 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3901 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3961 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4021 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4081 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4141 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4201 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     4261 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4321 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4381 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4441 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4501 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4561 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4621 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4681 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4741 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4801 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4861 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4921 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4981 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     5041 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     5101 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     5161 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     5221 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     5281 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     5341 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5401 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5461 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5521 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5581 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5641 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5701 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5761 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5821 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5881 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5941 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6001 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6061 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6121 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6181 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6241 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6301 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac c
//
