LOCUS       sequence-497009-        5644 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  Vector for protein expression of 2x StrepII-tagged Arabidopsis 
            heterotrimeric G protein GPA1-Q222L (""constitutively active"" 
            mutant) in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5644)
  AUTHORS   Gookin TE, Chakravorty D, Assmann SM
  TITLE     Influence of expression and purification protocols on Galpha 
            biochemical activity: kinetics of plant and mammalian G protein 
            cycles.
  JOURNAL   Front Mol Biosci. 2025 Apr 7;12:1513660. doi: 
            10.3389/fmolb.2025.1513660. eCollection 2025.
  PUBMED    40260404
REFERENCE   2  (bases 1 to 5644)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 13, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front Mol 
            Biosci."; date: "2025-04-7"; volume: "12:1513660. doi"; pages: " 
            10.3389/fmolb.2025.1513660. eCollection 2025"
COMMENT     Sequence Label: pSTTa-GPA1-Q222L
FEATURES             Location/Qualifiers
     source          1..5644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(180..185,186..201,202..208)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    216..232
                     /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)."
     CDS             273..296
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             330..353
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             363..380
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             387..407
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1593..1616
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1695..1717)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1862..1880)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1944..2048
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2049..2117,2118..2909)
                     /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(2267..2286)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3080..3668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3569..3588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3822..3839
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3912..3989
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3990..5072
                     /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     complement(4009..4028)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5121..5126,5127..5144,5145..5151)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5159..5175
                     /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)."
     primer_bind     5183..5199
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5183..5199
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5195..5644,1..78)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5215..5232)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5215..5231)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5224..5246)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta
       61 tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct
      121 ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt
      181 tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac
      241 aggaaacagt attcatgagc gctagcagcg cgtggagcca cccgcagttc gaaaagagcg
      301 gtggctcagg taccagtggc ggttcagcct ggagccatcc gcaattcgag aaggtcggca
      361 gcctggttcc gcgtggttct gctggtgaaa acctgtactt ccagggtggt tcaggatcca
      421 tgggcttact ctgcagtaga agtcgacatc atactgaaga tactgatgag aatacacagg
      481 ctgctgaaat cgaaagacgg atagagcaag aagcaaaggc tgaaaagcat attcggaagc
      541 ttttgctact tggtgctggg gaatctggaa aatctacaat ttttaagcag ataaaacttc
      601 tattccaaac gggatttgat gaaggagaac taaagagcta tgttccagtc attcatgcca
      661 atgtctatca gactataaaa ttattgcatg atggaacaaa ggagtttgct caaaatgaaa
      721 cagattctgc taaatatatg ttatcttctg aaagtattgc aattggggag aaactatctg
      781 agattggtgg taggttagac tatccacgtc ttaccaagga catcgctgag ggaatagaaa
      841 cactatggaa ggatcctgca atccaggaaa cttgtgctcg tggtaatgag cttcaggttc
      901 ctgattgtac gaaatatctg atggagaact tgaagagact atcagatata aattatattc
      961 caactaagga ggatgtactt tatgcaagag ttcgcacaac tggtgtcgtg gaaatacagt
     1021 tcagccctgt gggagagaat aaaaaaagtg gtgaagtgta ccgattgttt gacgtgggtg
     1081 gactgagaaa tgagaggagg aaatggattc atctgtttga aggtgtaaca gctgtgatat
     1141 tttgtgctgc catcagcgag tacgaccaaa cgctctttga ggacgagcag aaaaacagga
     1201 tgatggagac caaggaatta ttcgactggg tcctgaaaca accctgtttt gagaaaacat
     1261 ccttcatgct gttcttgaac aagttcgaca tatttgagaa gaaagttctt gacgttccgt
     1321 tgaacgtttg cgagtggttc agagattacc aaccagtttc aagtgggaaa caagagattg
     1381 agcatgcata cgagtttgtg aagaagaagt ttgaggagtt atattaccag aacacggcgc
     1441 cggatagagt ggacagggta ttcaaaatct acaggacgac ggctttggac cagaagcttg
     1501 taaagaaaac gttcaagctc gtagatgaga cactaagaag gagaaattta ctggaggctg
     1561 gccttttatg atccggagcg agcaccggtg cagattataa ggatgatgat gataagtaac
     1621 tgagtaagcg atcgcagaat tcatcgtgac tgactgacga tctgcctcgc gcgtttcggt
     1681 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     1741 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     1801 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag
     1861 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagcag
     1921 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     1981 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2041 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2101 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2161 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2221 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2281 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2341 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2401 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2461 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2521 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     2581 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     2641 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     2701 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     2761 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     2821 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     2881 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     2941 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3001 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3061 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3121 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3181 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3241 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3301 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3361 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3421 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3481 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3541 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3601 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3661 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3721 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3781 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3841 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3901 gcataaattc cgacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     3961 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     4021 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     4081 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     4141 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     4201 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     4261 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     4321 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     4381 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     4441 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     4501 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     4561 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     4621 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     4681 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     4741 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     4801 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     4861 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     4921 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     4981 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5041 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     5101 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     5161 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgg attcactggc
     5221 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     5281 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     5341 ccaacagttg cgcagcctga atggcgaatg gcgctttgcc tggtttccgg caccagaagc
     5401 ggtgccggaa agctggctgg agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc
     5461 aaactggcag atgcacggtt acgatgcgcc catctacacc aacgtgacct atcccattac
     5521 ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt tgttactcgc tcacatttaa
     5581 tgttgatgaa agctggctac aggaaggcca gacgcgaatt atttttgatg gcgttggaat
     5641 tagc
//
