LOCUS       sequence-321535-        7637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of proteins with a C-terminal thermostable GFP 
            (TGP) tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7637)
  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 7637)
  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: pBTSG
FEATURES             Location/Qualifiers
     source          1..7637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      102..556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(189..208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     398..419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        582..686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(687..755,756..1547)
                     /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     complement(905..924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1718..2306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2207..2226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2460..2477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2611..2835
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2902..3435)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3624..3629,3630..3646,3647..3652))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4169..4217
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4242..4261
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(4353..4462)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        4507..4598
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     4523..4540
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     enhancer        4778..5157
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5158..5361
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5312..5332
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5358..5382
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          5497..5629
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5612..5631
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      5689..5698
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5737..5760
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6454..6480
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     misc_feature    6506..7094
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6559..6579)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6977..6988)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7227..7361
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7264..7283)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7318..7337
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7390..7555
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga
       61 ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
      121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      301 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      361 ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga
      421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
      481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
      541 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      601 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
      661 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
      721 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
      781 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
      841 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
      901 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
      961 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1021 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1081 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1141 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1201 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     1261 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1321 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1381 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     1441 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1501 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     1561 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     1681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1861 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2101 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2161 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2221 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2281 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2341 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2401 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2461 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2521 tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg
     2581 agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa
     2641 caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga
     2701 aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact
     2761 cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg
     2821 gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc
     2881 gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac
     2941 ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc
     3001 ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag
     3061 cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat
     3121 ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc
     3181 ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta
     3241 atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag
     3301 atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat
     3361 gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt
     3421 gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg
     3481 cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga
     3541 aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg
     3601 atcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc
     3661 gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg
     3721 aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg
     3781 cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc
     3841 aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag
     3901 tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct
     3961 atagttctag tggttggcta cagctttgtt taaacaaagc tggctatggc agggcttgcc
     4021 gccccgacgt tggctgcgag ccctgggcct tcacccgaac ttgggggttg gggtggggaa
     4081 aaggaagaaa cgcgggcgta ttggtcccaa tggggtctcg gtggggtatc gacagagtgc
     4141 cagccctggg accgaacccc gcgtttatga acaaacgacc caacacccgt gcgttttatt
     4201 ctgtcttttt attgccgtca tagcgcgggt tccttccggt attgtctcct tccgtgtttc
     4261 agttagcctc ccccatctcc cgataccgca tgctatgcat cagctgctag caccatggct
     4321 cgagatcccg ggtgatcaag tcttcgtcga gtgattgtaa ataaaatgta atttacagta
     4381 tagtatttta attaatatac aaatgatttg ataataattc ttatttaact ataatatatt
     4441 gtgttgggtt gaattaaagg tccgtatact agtatcgatt cgcgacctac tccggaatat
     4501 taatagatca tggagataat taaaatgata accatctcgc aaataaataa gtattttact
     4561 gttttcgtaa cagttttgta ataaaaaaac ctataaatat tccggattat tcataccgtc
     4621 ccaccatcgg gcgcgaatcc tcaatattgg ccattagcca tattattcat tggttatata
     4681 gcataaatca atattggcta ttggccattg catacgttgt atctatatca taatatgtac
     4741 atttatattg gctcatgtcc aatatgaccg ccatgttggc attgattatt gactagttat
     4801 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     4861 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     4921 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     4981 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg
     5041 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     5101 ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     5161 atgcggtttt ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca
     5221 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     5281 ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg
     5341 gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcactagaag ctttattgcg
     5401 gtagtttatc acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac
     5461 ttaagctgca gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag
     5521 gtttaaggag accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga
     5581 taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc
     5641 cagttcaatt acagctctta aggaatcccg gtccgaagcg cgcgggccgc caccatggga
     5701 tccaagctta gcggccgcga attcctcgag ggtaccctgg aagtgctgtt tcagggcccg
     5761 ggcggtagtg gaggttccgc gagtgtcatt aagccggaga tgaaaattaa gttgcgtatg
     5821 gagggggccg tgaatgggca caaattcgtt attgaagggg aggggattgg aaagccgtat
     5881 gagggtacgc aaaccttgga cttaacagta gaagaagggg caccattgcc tttttcttat
     5941 gatattttga ctccggcgtt ccaatacggg aaccgtgctt tcacaaagta tcccgaagat
     6001 atccctgact atttcaagca agcatttccc gagggttaca gttgggagag aagtatgacc
     6061 tatgaggatc aaggcatatg tattgctacc tctgacatta ctatggaggg agactgcttc
     6121 ttctatgaga ttcgtttcga tggaacaaac tttcctccta acgggccagt gatgcagaaa
     6181 aagacgttga agtgggaacc gtccacagaa aaaatgtatg tcgaggacgg cgttctaaag
     6241 ggagacgtgg aaatggcctt gctattggaa gggggtggac actatcgttg tgatttcaaa
     6301 acgacctaca aggccaaaaa agatgtgaga ctaccggacg cccacgaggt tgaccatcgt
     6361 attgaaattt taagtcacga caaggactat aataaagtaa gactgtacga acacgcggaa
     6421 gcacgttaca gcggaggtgg aagtggtggt ggccatcacc atcaccatca ccatcaccat
     6481 taaagcctgc agtctcgaca agcttaatca acctctggat tacaaaattt gtgaaagatt
     6541 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     6601 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     6661 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     6721 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     6781 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     6841 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     6901 gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     6961 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     7021 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     7081 ggccgcctcc ccgcctgaag cttgtcgaga agtactagag gatcataatc agccatacca
     7141 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     7201 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     7261 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     7321 gtttgtccaa actcatcaat gtatcttatc atgtctggat ctgatcactg cttgagccta
     7381 ggagatccga accagataag tgaaatctag ttccaaacta ttttgtcatt tttaattttc
     7441 gtattagctt acgacgctac acccagttcc catctatttt gtcactcttc cctaaataat
     7501 ccttaaaaac tccatttcca cccctcccag ttcccaacta ttttgtccgc ccacagcggg
     7561 gcatttttct tcctgttatg tttttaatca aacatcctgc caactccatg tgacaaaccg
     7621 tcatcttcgg ctacttt
//
