LOCUS       sequence-321533-        5988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of proteins with a C-terminal thermostable GFP (TGP) tag 
            in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5988)
  AUTHORS   Cai H, Yao H, Li T, Hutter CAJ, Li Y, Tang Y, Seeger MA, Li D
  TITLE     An improved fluorescent tag and its nanobodies for membrane protein 
            expression, stability assay, and purification.
  JOURNAL   Commun Biol. 2020 Dec 10;3(1):753. doi: 10.1038/s42003-020-01478-z.
  PUBMED    33303987
REFERENCE   2  (bases 1 to 5988)
  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: "Commun 
            Biol."; date: "2020-12-10"; volume: "3(1):753. doi"; pages: " 
            10.1038/s42003-020-01478-z"
COMMENT     Sequence Label: pETSG
FEATURES             Location/Qualifiers
     source          1..5988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(88..107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(548..1363)
                     /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(548..1363)
                     /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     1271..1290
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1355..1374
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1485..2073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1974..1993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2227..2244
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2259..2398
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2324..2343)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2484..2506
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2500..2691)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3500..4582)
                     /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     4544..4563
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4583..4660)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4866..4885
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4969..4988
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4969..4987
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4988..5012
                     /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             5027..5049
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5098..5121
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5815..5838
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(5891..5909)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5905..5952
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
      541 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
      601 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
      661 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
      721 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
      781 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
      841 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
      901 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
      961 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     1021 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     1081 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     1141 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     1201 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     1261 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     1321 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     1381 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     1441 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1501 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1561 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1621 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1681 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1741 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1801 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1861 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1921 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1981 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2041 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2101 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     2161 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     2221 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     2281 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     2341 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     2401 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2461 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2521 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     2581 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     2641 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     2701 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     2761 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     2821 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     2881 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     2941 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     3001 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     3061 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     3121 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     3181 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     3241 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     3301 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     3361 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     3421 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     3481 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3541 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     3601 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     3661 agagttgcag caagcggtcc acgctggttt gccccagcag gcggaaatcc tgtttgatgg
     3721 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     3781 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     3841 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     3901 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     3961 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     4021 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     4081 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     4141 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     4201 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     4261 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     4321 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     4381 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     4441 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     4501 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     4561 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     4621 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     4681 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     4741 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     4801 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     4861 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     4921 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     4981 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     5041 agaaggagat ataccatggg atccaagctt agcggccgcg aattcctcga gggtaccctg
     5101 gaagtgctgt ttcagggccc gggcggtagt ggaggttccg cgagtgtcat taagccggag
     5161 atgaaaatta agttgcgtat ggagggggcc gtgaatgggc acaaattcgt tattgaaggg
     5221 gaggggattg gaaagccgta tgagggtacg caaaccttgg acttaacagt agaagaaggg
     5281 gcaccattgc ctttttctta tgatattttg actccggcgt tccaatacgg gaaccgtgct
     5341 ttcacaaagt atcccgaaga tatccctgac tatttcaagc aagcatttcc cgagggttac
     5401 agttgggaga gaagtatgac ctatgaggat caaggcatat gtattgctac ctctgacatt
     5461 actatggagg gagactgctt cttctatgag attcgtttcg atggaacaaa ctttcctcct
     5521 aacgggccag tgatgcagaa aaagacgttg aagtgggaac cgtccacaga aaaaatgtat
     5581 gtcgaggacg gcgttctaaa gggagacgtg gaaatggcct tgctattgga agggggtgga
     5641 cactatcgtt gtgatttcaa aacgacctac aaggccaaaa aagatgtgag actaccggac
     5701 gcccacgagg ttgaccatcg tattgaaatt ttaagtcacg acaaggacta taataaagta
     5761 agactgtacg aacacgcgga agcacgttac agcggaggtg gaagtggtgg tggccatcat
     5821 catcaccacc accaccattg agatccggct gctaacaaag cccgaaagga agctgagttg
     5881 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     5941 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatggg
//
