LOCUS       sequence-213407-        7520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of glutaminase isoform KGA (wild type) with V5 
            and 6xHis tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7520)
  AUTHORS   Cassago A, Ferreira AP, Ferreira IM, Fornezari C, Gomes ER, Greene 
            KS, Pereira HM, Garratt RC, Dias SM, Ambrosio AL
  TITLE     Mitochondrial localization and structure-based phosphate activation 
            mechanism of Glutaminase C with implications for cancer metabolism.
  JOURNAL   Proc Natl Acad Sci U S A. 2012 Jan 24;109(4):1092-7. doi: 
            10.1073/pnas.1112495109. Epub 2012 Jan 6.
  PUBMED    22228304
REFERENCE   2  (bases 1 to 7520)
  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: 
            "10.1073/pnas.1112495109"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2012-01-24- 24"; volume: "109"; issue: "4"; pages: "1092-7"
COMMENT     Sequence Label: pcDNA3.1D V5-His-TOPO-KGA.wt
FEATURES             Location/Qualifiers
     source          1..7520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2784..2825
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2835..2852
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2875..2892)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2881..3105
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3151..3579
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3238..3257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3448..3469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3588..3608)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3593..3922
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3773..3908
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3835..3854
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3989..4783
                     /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(4043..4062)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4653..4672
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4959..5080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4996..5015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5050..5069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5129..5145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5129..5145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5142..5164)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5153..5169
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5177..5183,5184..5201,5202..5207))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5222..5243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5360..5377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5531..6119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5611..5630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6290..7081,7082..7150))
                     /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     6913..6932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7151..7255)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7330..7349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagttaag cttggtaccg agctcggatc cagtaccctt caccatgatg cggctgcgag
      721 gctcggggat gctgcgggac ctgctcctgc ggtcgcccgc cggcgtgagc gcgactctgc
      781 ggcgggcaca gcccttggtc accctgtgcc ggcgtccccg aggcggggga cggccggccg
      841 cgggcccggc tgccgccgcg cgactccacc cgtggtgggg cgggggcggc tggccggcgg
      901 agcccctcgc gcggggcctg tccagctctc cttcggagat cttgcaggag ctgggcaagg
      961 ggagcacgca tccgcagccc ggggtgtcgc cacccgctgc cccggcggcg cccggcccca
     1021 aggacggccc cggggagacg gacgcgtttg gcaacagcga gggcaaagag ctggtggcct
     1081 caggtgaaaa taaaataaaa cagggtctgt tacctagctt ggaagatttg ctgttctata
     1141 caattgctga aggacaagag aaaatacctg ttcataaatt tattacagca ctcaaatcta
     1201 caggattgcg aacgtctgat cccaggttga aagagtgtat ggatatgtta agattaactc
     1261 ttcaaacaac atcagatggt gtcatgctag acaaagatct ttttaaaaaa tgtgttcaga
     1321 gcaacattgt tttgttgaca caagcattta gaagaaagtt tgtgattcct gactttatgt
     1381 cttttacctc acacattgat gagttatatg aaagtgctaa aaagcagtct ggaggaaagg
     1441 ttgcagatta tattcctcaa ctggccaaat tcagtcccga tttgtggggt gtgtctgttt
     1501 gtacagtaga tggacagagg cattctactg gagataccaa agttcccttc tgtcttcagt
     1561 cctgtgtaaa acctttgaaa tatgccattg ctgttaatga tcttggaact gaatatgtgc
     1621 atcgatatgt tggaaaagag ccgagtggac taagattcaa caaactattt ttgaatgaag
     1681 atgataaacc acataatcct atggtaaatg ctggagcaat tgttgtgact tcactaataa
     1741 agcaaggagt aaataatgct gaaaaatttg actatgtcat gcagtttttg aataagatgg
     1801 ctggtaatga atatgttgga ttcagtaatg caacgtttca gtctgaaaga gaaagtggag
     1861 atcgaaattt tgcaatagga tattacttaa aagaaaagaa gtgttttcca gaaggcacag
     1921 acatggttgg tatattagac ttctacttcc agctgtgctc cattgaagtg acttgtgaat
     1981 cagccagtgt gatggctgcg acactggcta atggtggttt ctgcccaatt actggtgaaa
     2041 gagtactgag ccctgaagca gttcgaaata cattgagttt gatgcattcc tgtggcatgt
     2101 atgacttctc agggcagttt gctttccatg ttggtcttcc tgcaaaatct ggagttgctg
     2161 ggggcattct tttagttgtc cccaatgtta tgggtatgat gtgctggtct cctcctctgg
     2221 ataagatggg caacagtgtt aagggaattc acttttgtca cgatcttgtt tctctgtgta
     2281 atttccataa ctatgataat ttgagacact ttgcaaaaaa acttgatcct cgaagagaag
     2341 gtggtgatca aagggtaaag tcagtgataa atcttttgtt tgctgcatat actggagatg
     2401 tgtctgcact tcgaagattt gctttgtcag ctatggacat ggaacagcgg gactatgatt
     2461 ctagaacagc actccatgta gctgctgcag agggtcatgt tgaagttgtt aaatttttgc
     2521 tggaagcctg caaagtaaac cctttcccca aggacaggtg gaataacact cccatggatg
     2581 aagcactgca ctttggacac catgatgtat ttaaaattct ccaagaatac caagtccagt
     2641 acacacctca aggagattct gacaacggga aggaaaatca aaccgtccat aagaatcttg
     2701 atggattgtt gaagggtcaa gacaattctg cagatatcca gcacagtggc ggccgctcga
     2761 gtctagaggg cccgcggttc gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt
     2821 ctacgcgtac cggtcatcat caccatcacc attgagttta aacccgctga tcagcctcga
     2881 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2941 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3001 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3061 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3121 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3181 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3241 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3301 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3361 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3421 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3481 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3541 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     3601 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3661 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3721 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3781 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3841 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     3901 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     3961 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     4021 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4081 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4141 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     4201 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4261 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4321 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4381 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     4441 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     4501 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     4561 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     4621 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4681 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4741 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4801 gcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4861 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4921 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4981 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5041 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5101 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5161 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5221 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5281 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5341 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5401 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5461 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5521 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5581 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5641 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5701 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5761 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagaccgac cgctgcgcct
     5821 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5881 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5941 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6001 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6061 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6121 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6181 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6241 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6301 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6361 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6421 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6481 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6541 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6601 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6661 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6721 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6781 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6841 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6901 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6961 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7021 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7081 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7141 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7201 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7261 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     7321 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     7381 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     7441 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     7501 acgggccaga tatacgcgtt
//
