LOCUS       sequence-256582-        7095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses human PPAT protein with a C-terminal 2XStrep affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7095)
  AUTHORS   Liu C, Knudsen GM, Pedley AM, He J, Johnson JL, Yaron TM, Cantley 
            LC, Benkovic SJ
  TITLE     Mapping Post-Translational Modifications of de Novo Purine 
            Biosynthetic Enzymes: Implications for Pathway Regulation.
  JOURNAL   J Proteome Res. 2019 May 3;18(5):2078-2087. doi: 
            10.1021/acs.jproteome.8b00969. Epub 2019 Apr 18.
  PUBMED    30964683
REFERENCE   2  (bases 1 to 7095)
  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: 
            "10.1021/acs.jproteome.8b00969"; journalName: "J Proteome Res"; 
            date: "2019-05-3- 3"; volume: "18"; issue: "5"; pages: "2078-2087"
COMMENT     Sequence Label: pPPAT-2XStrep
FEATURES             Location/Qualifiers
     source          1..7095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        143..522
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        523..734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     685..705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     731..755
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          895..1027
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1010..1029
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1072..1090
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2662..2667,2668..2691,2692..2727,2728..2751)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     polyA_signal    2804..2925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2815..2834)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2869..2888
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3111..3566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3198..3217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3408..3429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3683..4040
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3683..3874
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3706..3726)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3891..4026
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4091..4885
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4145..4164)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4755..4774
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4949..4997
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5038..5057)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5157..5179
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5217..5235)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5303..5407
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5408..5476,5477..6268)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5626..5645)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6439..7027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6928..6947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatcttcaat attggccatt agccatatta ttcattggtt atatagcata aatcaatatt
       61 ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta tattggctca
      121 tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt
      181 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      241 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      301 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      361 actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc
      421 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct
      481 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      541 tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      601 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      661 aactgcgatc gcccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      721 atataagcag agctcgttta gtgaaccgtc agatcactag aagctttatt gcggtagttt
      781 atcacagtta aattgctaac gcagtcagtg cttctgacac aacagtctcg aacttaagct
      841 gcagtgactc tcttaaggta gccttgcaga agttggtcgt gaggcactgg gcaggtaagt
      901 atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc gagacagaga
      961 agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt tgcctttctc
     1021 tccacaggtg tccactccca gttcaattac agctcttaag gctagagtac ttaatacgac
     1081 tcactatagg ctagcctcga gatggagctg gaggagttgg ggatccgaga ggaatgtggc
     1141 gtgttcgggt gcatcgcctc aggagagtgg cccacgcagc tggatgtacc gcatgtgatc
     1201 actctgggac tcgtggggct gcagcaccgg ggtcaggaga gtgctggtat tgtgactagt
     1261 gatgggagtt ccgtgccaac attcaaatca cacaagggaa tgggtcttgt aaatcacgtc
     1321 tttactgaag acaatttgaa aaaattatat gtttcaaatc ttggaattgg acacaccagg
     1381 tatgccacca caggaaaatg tgaactagaa aattgtcagc ccttcgttgt tgaaacactt
     1441 catgggaaga tagctgtggc acataatggc gaattggtaa atgctgctcg attaaggaaa
     1501 aagcttctgc gtcatggtat tggtctgtct acaagttctg atagtgaaat gattacccag
     1561 ttactggcgt atacccctcc tcaggaacaa gatgacaccc cagactgggt agccaggatt
     1621 aaaaacttga tgaaggaagc acccacagca tactccctgc ttataatgca cagagatgtt
     1681 atttatgcag tacgagatcc ttatggaaat cgtcccttat gcattggtcg tcttattcca
     1741 gtgtctgata taaatgacaa agagaaaaaa acatcagaaa cagaaggatg ggtggtgtct
     1801 tcagaatctt gtagcttctt atctattggt gcaagatatt accgtgaagt cttgcctgga
     1861 gaaattgtgg aaatatccag acacaatgtc caaactcttg atattatatc aaggtctgaa
     1921 ggaaacccag tggctttttg tatctttgaa tatgtttatt ttgcaagacc agacagtatg
     1981 ttcgaagacc aaatggttta tacagtaaga taccgttgtg gccagcagct agcgattgaa
     2041 gcacctgtgg atgcagattt ggttagcact gttccagaat ctgctacgcc tgctgctctt
     2101 gcttacgcag gaaagtgtgg acttccatat gtggaggtgc tgtgtaaaaa ccggtatgta
     2161 gggagaacct tcattcagcc aaacatgagg ttaagacaac ttggtgttgc aaaaaaattt
     2221 ggagtattgt cagacaactt taaaggcaaa agaattgttc ttgtagatga ttcaattgtc
     2281 agaggcaata ccatctcacc tataataaaa ctgctcaaag aatctggtgc aaaagaggta
     2341 cacattcgag tagcttcacc accaattaaa tatccatgct tcatgggaat aaacattcct
     2401 acaaaagaag agctcattgc caataaacca gaatttgatc accttgcaga atatctagga
     2461 gcaaacagtg ttgtgtatct gtcagtagaa ggactggttt catctgtaca agaagggata
     2521 aagtttaaaa aacagaaaga gaaaaagcac gatattatga tccaagaaaa tggaaatggt
     2581 ctggaatgtt ttgaaaagag tggtcattgt acagcttgtc tcactggaaa atatcctgta
     2641 gaattagaat gggtacccgg gagcgcttgg agccacccgc agttcgaaaa aggtggaggt
     2701 tctggcggtg gatcgggagg ttcagcgtgg agccacccgc agttcgagaa ataaagcgcg
     2761 gccgcttccc tttagtgagg gttaatgctt cgagcagaca tgataagata cattgatgag
     2821 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     2881 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     2941 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     3001 ctctacaaat gtggtaaaat ccgataagga tcgatccggg ctggcgtaat agcgaagagg
     3061 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg
     3121 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3181 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3241 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3301 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3361 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3421 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3481 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3541 taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc
     3601 ggtatttcac accgcatacg cggatctgcg cagcaccatg gcctgaaata acctctgaaa
     3661 gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     3721 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3781 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3841 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3901 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3961 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4021 gaggcctagg cttttgcaaa aagcttgatt cttctgacac aacagtctcg aacttaaggc
     4081 tagagccacc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4141 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4201 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4261 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4321 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4381 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4441 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4501 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4561 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     4621 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4681 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4741 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4801 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4861 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4921 acgcccaacc tgccatcacg atggccgcaa taaaatatct ttattttcat tacatctgtg
     4981 tgttggtttt ttgtgtgaat cgatagcgat aaggatccgc gtatggtgca ctctcagtac
     5041 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     5101 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5161 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     5221 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5281 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5341 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5401 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5461 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5521 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5581 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5641 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5701 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5761 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5821 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5881 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5941 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6001 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6061 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6121 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6181 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6241 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6301 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6361 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6421 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6481 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6541 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6601 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6661 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6721 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6781 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6841 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6901 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6961 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7021 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7081 tcacatggct cgaca
//
