LOCUS       sequence-220870-        4640 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Cherry and GFP-SidM-P4M domain (2 copies).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4640)
  AUTHORS   Botero S, Chiaroni-Clarke R, Simon SM
  TITLE     Escherichia coli as a platform for the study of phosphoinositide 
            biology.
  JOURNAL   Sci Adv. 2019 Mar 27;5(3):eaat4872. doi: 10.1126/sciadv.aat4872. 
            eCollection 2019 Mar.
  PUBMED    30944849
REFERENCE   2  (bases 1 to 4640)
  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: "Sci Adv."; 
            date: "2019-03-27"; volume: "5(3):eaat4872. doi"; pages: " 
            10.1126/sciadv.aat4872. eCollection 2019 Mar"
COMMENT     Sequence Label: pClodAcytCh-GFP-P4M
FEATURES             Location/Qualifiers
     source          1..4640
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..69,70..861)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(219..238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     RBS             1174..1196
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1727..1738
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     rep_origin      complement(1992..2730)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             join(2920..2922,2923..2925,2926..3636)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2920..2922,2923..2925,2926..3636)
                     /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(2965..2986)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3226..3245)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3573..3594
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3700..4005
                     /codon_start=1
                     /product="high-affinity phosphatidylinositol 4-phosphate 
                     binding module of SidM (DrrA) from Legionella pneumophila 
                     (Hammond et al., 2014)"
                     /label=P4M
                     /translation="TASTENFKNVKEKYQQMRGDALKTEILADFKDKLAEATDEQSLKQ
                     IVAELKSKDEYRILAKGQGLTTQLLGLKTSSVSSFEKMVEETRESIKSQERQTIKIK"
     CDS             4051..4356
                     /codon_start=1
                     /product="high-affinity phosphatidylinositol 4-phosphate 
                     binding module of SidM (DrrA) from Legionella pneumophila 
                     (Hammond et al., 2014)"
                     /label=P4M
                     /translation="TASTENFKNVKEKYQQMRGDALKTEILADFKDKLAEATDEQSLKQ
                     IVAELKSKDEYRILAKGQGLTTQLLGLKTSSVSSFEKMVEETRESIKSQERQTIKIK"
     primer_bind     complement(4450..4468)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4536..4640
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
       61 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      121 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      181 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      241 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      301 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      361 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
      421 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
      481 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
      541 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
      601 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
      661 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
      721 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
      781 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
      841 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
      901 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
      961 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccac catggacaca
     1021 gctaacacca cgtcgtccct atctgctgcc ctaggtctat gagtggttgc tggataactt
     1081 tacgggcatg cataaggctc gtataatata ttcagggaga ccacaacggt ttccctctac
     1141 aaataatttt gtttaacttt gagggacaat aattttgttt aactttaaga aggagatata
     1201 cattactagt atcgatggtc tcaaagggtg aagaggacaa catggcaata attaaagagt
     1261 tcatgagatt taaagtccat atggagggtt cagtcaacgg acacgagttc gagattgagg
     1321 gtgagggaga gggaaggcca tacgagggaa ctcagactgc aaagttgaag gtcacaaaag
     1381 gtggaccttt accattcgca tgggacatat tatcaccaca gtttatgtat ggttctaagg
     1441 catacgtcaa gcacccagct gatattccag actacttgaa gttgtctttt cctgaaggtt
     1501 tcaaatggga gagggtcatg aacttcgagg acggaggagt cgtcactgtc actcaggact
     1561 catctttgca ggatggagag tttatataca aagttaaatt gaggggaaca aacttcccat
     1621 cagacggacc agtcatgcaa aagaagacta tgggttggga ggcttcttca gagaggatgt
     1681 acccagagga cggagctttg aagggtgaga taaaacaaag gttgaaattg aaggacggtg
     1741 gtcactacga cgctgaagtt aagacaacat acaaggctaa gaagcctgtc caattgcctg
     1801 gtgcttataa cgtcaacatt aaattggaca ttacttctca caacgaggat tacactattg
     1861 tcgagcaata cgagagggct gagggaagac actctactgg aggtatggac gagttgtaca
     1921 aataataagt cgacgttaat aacaggcctg ctggtaatcg caggcctttt tatttttttt
     1981 tttaagatct agcgctgcgg acacatacaa agttgcccac agattccgtg gataagcagg
     2041 ggactaacat gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg
     2101 ccctcctgcc agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag
     2161 gctcaaccat gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt
     2221 ttccggcggg tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc
     2281 tgttccgcct ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat
     2341 ctcggctcgg tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag
     2401 cccgactgct gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt
     2461 aaaacgccac tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa
     2521 acacgcggtt gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg
     2581 ttgctgtgct ctgcgaaagc cagttaccac ggttaagcag ttccccaact gacttaacct
     2641 tcgatcaaac cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga
     2701 aaaaaaggat ctcaagaaga tcctttgatc agcggccgcc acagctaaca ccacgtcgtc
     2761 cctatctgct gccctaggtc tatgagtggt tgctggataa ctttacgggc atgcataagg
     2821 ctcgtataat atattcaggg agaccacaac ggtttccctc tacaaataat tttgtttaac
     2881 tttcaattga aggagatata catactagta tcgggatcca tggtgagcaa gggcgaggag
     2941 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     3001 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     3061 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     3121 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     3181 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     3241 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     3301 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     3361 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     3421 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     3481 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     3541 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     3601 gccgggatca ctctcggcat ggacgagctg tacaagtccg gactcagatc tcgagctcaa
     3661 gcttcgaatt ctggtggtag tgcttccgga ctcagatcca cggcaagcac ggaaaacttt
     3721 aaaaatgtta aagaaaaata tcagcaaatg cgaggtgatg ctttaaaaac agaaatcctg
     3781 gctgatttca aggataaact ggctgaagct accgatgaac agagccttaa gcaaattgtt
     3841 gctgaattga aaagtaaaga tgaatataga attttggcta aggggcaagg gttaacaacc
     3901 cagcttttag ggttaaagac aagttcagtg tcttcatttg agaaaatggt tgaggaaaca
     3961 agagaaagta ttaagtctca agaaagacaa accattaaga taaaaggtgg tagtgctgtc
     4021 gacggtggta gtgcttccgg actcagatcc acggcaagca cggaaaactt taaaaatgtt
     4081 aaagaaaaat atcagcaaat gcgaggtgat gctttaaaaa cagaaatcct ggctgatttc
     4141 aaggataaac tggctgaagc taccgatgaa cagagcctta agcaaattgt tgctgaattg
     4201 aaaagtaaag atgaatatag aattttggct aaggggcaag ggttaacaac ccagctttta
     4261 gggttaaaga caagttcagt gtcttcattt gagaaaatgg ttgaggaaac aagagaaagt
     4321 attaagtctc aagaaagaca aaccattaag ataaaataat aaaccggttg atgagttaat
     4381 aacaggcctg ctggtaatcg caggcctttt tatttttttt tttgctagca aattcttgaa
     4441 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4501 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4561 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4621 aatattgaaa aaggaagagt
//
