LOCUS       sequence-497014-        4519 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  E. coli protein expression vector encoding 2x N-terminal StrepII 
            tags flanking an 11 residue flexible linker, thrombin and TEV 
            protease sites, and an optional C-terminal FLAG tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4519)
  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 4519)
  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
FEATURES             Location/Qualifiers
     source          1..4519
                     /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             468..491
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(570..592)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(737..755)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        819..923
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(924..992,993..1784)
                     /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(1142..1161)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1955..2543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2444..2463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2697..2714
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2787..2864
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2865..3947
                     /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(2884..2903)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3996..4001,4002..4019,4020..4026)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4034..4050
                     /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     4058..4074
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4058..4074
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4070..4519,1..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(4090..4107)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4090..4106)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4099..4121)
                     /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 tggggtccct ccgtagtcac gtgacgtccg gagcgagcac cggtgcagat tataaggatg
      481 atgatgataa gtaactgagt aagcgatcgc agaattcatc gtgactgact gacgatctgc
      541 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
      601 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
      661 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat
      721 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
      781 atggtttctt agcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
      841 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
      901 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
      961 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     1021 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     1081 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     1141 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     1201 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     1261 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     1321 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     1381 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     1441 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     1501 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     1561 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     1621 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     1681 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     1741 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     1801 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     1861 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     1921 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     1981 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2041 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2101 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2161 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2221 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2281 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2341 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2401 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2461 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2521 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2581 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     2641 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     2701 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     2761 gcggtatttc acaccgcata aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta
     2821 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     2881 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     2941 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     3001 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     3061 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     3121 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     3181 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     3241 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     3301 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     3361 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     3421 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     3481 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     3541 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     3601 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     3661 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     3721 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     3781 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     3841 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     3901 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     3961 aatgtaagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     4021 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     4081 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     4141 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     4201 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt
     4261 tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac
     4321 tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt
     4381 gacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta
     4441 ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt
     4501 tgatggcgtt ggaattagc
//
