LOCUS       sequence-476594-        6854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli succinate semialdehyde dehydrogenase (SSDH/gabD) 
            for recombinant protein purification. Contains a His6-tag on its 
            C-terminus for Ni2+ affinity chromatography..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6854)
  AUTHORS   Benidickson KH, Symonds KF, Snedden WA, Plaxton WC
  TITLE     Cost-effective production of Escherichia coli ""GABase"" for 
            spectrophotometric determination of gamma-aminobutyrate (GABA) 
            levels or glutamate decarboxylase activity.
  JOURNAL   Biol Methods Protoc. 2025 Jun 20;10(1):bpaf050. doi: 
            10.1093/biomethods/bpaf050. eCollection 2025.
  PUBMED    40656557
REFERENCE   2  (bases 1 to 6854)
  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: "Biol 
            Methods Protoc."; date: "2025-06-20"; volume: "10(1):bpaf050. doi"; 
            pages: " 10.1093/biomethods/bpaf050. eCollection 2025"
COMMENT     Sequence Label: pET30a-SSDH
FEATURES             Location/Qualifiers
     source          1..6854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3489..3508
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3489..3507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3508..3532
                     /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)."
     RBS             3547..3569
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3580..3597
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3607..3624
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3631..3675
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3691..3705
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             5203..5220
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5273..5291)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5287..5334
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5371..5826
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5458..5477)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5668..5689
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5918..6733)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5918..6733)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6641..6660
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6725..6744
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     3481 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     3541 aataattttg tttaacttta agaaggagat atacatatgc accatcatca tcatcattct
     3601 tctggtctgg tgccacgcgg ttctggtatg aaagaaaccg ctgctgctaa attcgaacgc
     3661 cagcacatgg acagcccaga tctgggtacc gacgacgacg acaaggccat ggctgatatc
     3721 ggatccatga aacttaacga cagtaaatta ttccgccagc aggcgttgat taacggggaa
     3781 tggctggacg ccaacaatgg cgaggtcatc gatgtcacca atccggcgaa cggcgacaag
     3841 ctgggtagcg tacccaaaat gggcgctgat gaaacccgcg ccgctatcga cgccgccaac
     3901 cgcgctctgc ccgcctggcg tgcgctcacc gccaaagaac gcgccaacat tctgcgcaac
     3961 tggttcaatt tgttgatgga gcatcaggac gatttagcgc gcctgatgac cctagaacag
     4021 ggcaaaccgc tggctgaagc gaaaggcgaa attagctacg ccgcctcctt tattgagtgg
     4081 tttgctgaag aaggcaaacg catttatggc gacaccattc ctggtcatca ggccgataag
     4141 cgcctgattg ttatcaagca gccgattggc gtcaccgcag ctatcacgcc gtggaacttc
     4201 ccggcggcga tgattacccg caaagccggt ccggcgctgg cggcaggctg caccatggtg
     4261 ctgaagcctg ccagtcagac gccgttctct gcgctggcgc tggcggagct ggcgattcgc
     4321 gcgggcattc cggctggggt atttaacgtg gtcaccggtt cggcgggcgc ggtgggtaac
     4381 gaactgacca gcaacccgct ggtgcgcaag ctgtcgttta ccggttcgac cgaaattggc
     4441 cgccagttaa tggaacagtg cgcgaaagac atcaaaaaag tgtcgctgga gctgggcggc
     4501 aacgcgccgt ttatcgtctt tgacgatgcc gacctcgaca aagccgtgga aggtgcgctg
     4561 tcctcgaaat tccgcaacgc cgggcaaacc tgcgtctgcg ccaaccgttt atacgtgcag
     4621 gacggcgtgt atgaccgctt tgccgaaaaa ttgcagcagg cggtgagcaa attgcacatc
     4681 ggcgacgggc tggagaaagg cgtcaccatc gggccgctga tcgatgaaaa agcggtagca
     4741 aaagtggaag agcatattgc cgatgcgctg gagaaaggtg cgcgtgtggt ttgcggcggt
     4801 aaagcggatg aacgcggcgg caacttcttc cagccgacca ttctggtgga cgttccagcc
     4861 aacgccaaag tgtcgaaaga agagacgttc ggccccctcg ccccactgtt ccgttttaaa
     4921 gatgaagccg atgtgattgc gcaagccaat gacaccgagt ttggccttgc cgcctatttc
     4981 tacgcccgtg atttaagccg cgtcttccgc gtgggcgaag cgctggagta cggcatcgtc
     5041 ggcatcaata ccggcatcat ttccaatgaa gtggccccgt tcggcggcat taaagcctcg
     5101 ggtctgggtc gtgaaggttc gaagtatggc atcgaagatt acttagaaat caaatatatg
     5161 tgcatcggtc tttaagtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac
     5221 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     5281 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     5341 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     5401 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5461 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5521 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5581 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5641 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5701 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5761 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     5821 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5881 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     5941 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     6001 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     6061 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     6121 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     6181 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     6241 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     6301 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     6361 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     6421 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     6481 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     6541 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     6601 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     6661 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     6721 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     6781 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6841 atcaaaggat cttc
//
