LOCUS       sequence-197925-        6261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human PPAT-EGFP fusion in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6261)
  AUTHORS   An S, Kumar R, Sheets ED, Benkovic SJ
  TITLE     Reversible compartmentalization of de novo purine biosynthetic 
            complexes in living cells.
  JOURNAL   Science. 2008 Apr 4;320(5872):103-6. doi: 10.1126/science.1152241.
  PUBMED    18388293
REFERENCE   2  (bases 1 to 6261)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2008-04-4- 4"; volume: "320"; issue: 
            "5872"; pages: "103-6"
COMMENT     Sequence Label: pPPAT-EGFP
FEATURES             Location/Qualifiers
     source          1..6261
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        54..357
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        358..561
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     512..532
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2194..2203
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2200..2202,2203..2205,2206..2919)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLK
                     ERVTAAGITLGMDELYK"
     primer_bind     complement(2245..2266)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2506..2525)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    3042..3163
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3079..3098)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3133..3152
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3170..3625)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3307..3328)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3519..3538
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3652..3756
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3758..4115
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3758..3949
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3781..3801)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3966..4101
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4150..4944
                     /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             4150..4944
                     /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(4204..4223)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4814..4833
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5132..5151)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5176..5223
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5552..6140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6041..6060
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
       61 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      121 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      181 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      241 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      301 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      361 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      421 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      481 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      541 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact
      601 cagatctcga gccatatgga gctggaggag ttggggatcc gagaggaatg tggcgtgttc
      661 gggtgcatcg cctcaggaga gtggcccacg cagctggatg taccgcatgt gatcactctg
      721 ggactcgtgg ggctgcagca ccggggtcag gagagtgctg gtattgtgac tagtgatggg
      781 agttcggtgc caacattcaa atcacacaag ggaatgggtc ttgtaaatca cgtctttact
      841 gaagacaatt tgaaaaaatt atatgtttca aatcttggaa ttggacacac caggtatgcc
      901 accacaggaa aatgtgaact agaaaattgt cagcccttcg ttgttgaaac acttcatggg
      961 aagatagctg tggcacataa tggcgaattg gtaaatgctg ctcgattaag gaaaaagctt
     1021 ctgcgtcatg gtattggtct gtctacaagt tctgatagtg aaatgattac ccagttactg
     1081 gcgtataccc ctcctcagga acaagatgac accccagact gggtagccag gattaaaaac
     1141 ttgatgaagg aagcacccac agcatactcc ctgcttataa tgcacagaga tgttatttat
     1201 gcagtacgag atccttatgg aaatcgtccc ttatgcattg gtcgtcttat tccagtgtct
     1261 gatataaatg acaaagagaa aaaaacatca gaaacagaag gatgggtggt gtcttcagaa
     1321 tcttgtagct tcttatctat tggtgcaaga tattaccgtg aagtcttgcc tggagaaatt
     1381 gtggaaatat ccagacacaa tgtccaaact cttgatatta tatcaaggtc tgaaggaaac
     1441 ccagtggctt tttgtatctt tgaatatgtt tattttgcaa gaccagacag tatgttcgaa
     1501 gaccaaatgg tttatacagt aagataccgt tgtggccagc agctagcgat tgaagcacct
     1561 gtggatgcag atttggttag cactgttcca gaatctgcta cgcctgctgc tcttgcttac
     1621 gcaggaaagt gtggacttcc atatgtggag gtgctgtgta aaaaccggta tgtagggaga
     1681 accttcattc agccaaacat gaggttaaga caacttggtg ttgcaaaaaa atttggagta
     1741 ttgtcagaca actttaaagg caaaagaatt gttcttgtag atgattcaat tgtcagaggc
     1801 aataccatct cacctataat aaaactgctc aaagaatctg gtgcaaaaga ggtacacatt
     1861 cgagtagctt caccaccaat taaatatcca tgcttcatgg gaataaacat tcctacaaaa
     1921 gaagagctca ttgccaataa accagaattt gatcaccttg cagaatatct aggagcaaac
     1981 agtgttgtgt atctgtcagt agaaggactg gtttcatctg tacaagaagg gataaagttt
     2041 aaaaaacaga aagagaaaaa gcacgatatt atgatccaag aaaatggaaa tggtctggaa
     2101 tgttttgaaa agagtggtca ttgtacagct tgtctcactg gaaaatatcc tgtagaatta
     2161 gaatgggtac cgcgggcccg ggatccaccg gtcgccacca tggtgagcaa gggcgaggag
     2221 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2281 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     2341 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2401 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2461 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2521 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2581 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2641 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2701 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2761 cccatcggcg acggtcccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     2821 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgaaggagcg cgtgaccgcc
     2881 gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca
     2941 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3001 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3061 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3121 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc
     3181 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3241 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3301 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3361 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3421 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3481 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3541 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3601 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3661 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3721 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     3781 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3841 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3901 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3961 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4021 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4081 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     4141 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4201 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4261 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4321 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4381 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4441 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4501 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4561 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4621 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     4681 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4741 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4801 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4861 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4921 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4981 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5041 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5101 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     5161 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     5221 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     5281 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     5341 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     5401 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5461 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5521 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5581 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5641 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5701 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5761 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5821 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5881 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5941 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6001 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6061 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6121 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6181 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6241 ttaccgccat gcattagtta t
//
