LOCUS       sequence-169849-        5975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET28a expressing codon optimized superfolder GFP with C-terminal 
            His6 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5975)
  AUTHORS   Peeler JC, Mehl RA
  TITLE     Site-specific incorporation of unnatural amino acids as probes for 
            protein conformational changes.
  JOURNAL   Methods Mol Biol. 2012;794:125-34. doi: 10.1007/978-1-61779-331-8_8.
  PUBMED    21956560
REFERENCE   2  (bases 1 to 5975)
  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: "Methods Mol
            Biol."; date: "2012"; volume: "794:125-34. doi"; pages: " 
            10.1007/978-1-61779-331-8_8"
COMMENT     Sequence Label: pET28a-sfGFP
FEATURES             Location/Qualifiers
     source          1..5975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(169..186)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(193..903,904..906,907..909))
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     RBS             916..938
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    953..977
                     /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     complement(977..996)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(978..996)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1080..1099)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1305..1382
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1383..2465
                     /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(1402..1421)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3274..3465
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3459..3481)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3567..3706
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3622..3641
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3721..3738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3892..4480)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3972..3991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4591..4610)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4602..5417
                     /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             4602..5417
                     /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     complement(4675..4694)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5509..5964)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5646..5667)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5858..5877
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagctttaat ggtgatgatg
      181 atggtggctg cctttataca gttcatccat accgtgggta atgcccgcgg cggtaacaaa
      241 ttccagcagc accatatgat cacgtttttc attcggatct ttgctcagaa cgctctgggt
      301 gctcagataa tgattatccg gcagcagcac cgggccatca ccaatcgggg tattctgctg
      361 ataatgatcc gccagctgca cgctaccatc ttccacgttg tgacggattt taaagttcgc
      421 tttgatgcca tttttctgtt tatcggcggt aatatacaca ttatggctgt tgaaattata
      481 ttccagttta tgacccagaa tgttgccatc ttctttaaaa tcaatacctt tcagttcaat
      541 gcggttcacc agggtatcgc cttcaaattt aacttccgca cgggttttat aggtgccatc
      601 atctttgaag ctaatggtac gttcctgcac atagccttcc ggcatcgcgc ttttaaagaa
      661 atcatggcgt ttcatatgat ccggatagcg gctaaagcac tgaacgccat aggtcagggt
      721 ggtcaccagg gtcggccacg gaaccggcag tttaccggtg gtgcaaataa atttcagggt
      781 cagtttaccg ttggtcgcat caccttcgcc ttcgccacga acgctaaatt tatggccatt
      841 cacatcacca tccagttcca ccagaatcgg cacaacgccg gtaaacagtt cttcaccttt
      901 gctaaccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
      961 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat
     1021 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
     1081 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
     1141 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
     1201 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     1261 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
     1321 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     1381 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     1441 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     1501 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     1561 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     1621 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     1681 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     1741 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     1801 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     1861 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     1921 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     1981 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     2041 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     2101 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     2161 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     2221 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     2281 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     2341 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     2401 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2461 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
     2521 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     2581 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     2641 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
     2701 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
     2761 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg
     2821 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     2881 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     2941 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     3001 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     3061 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     3121 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     3181 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     3241 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     3301 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     3361 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     3421 cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3481 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3541 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     3601 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg
     3661 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     3721 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3781 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3841 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3901 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3961 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4021 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4081 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4141 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4201 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4261 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4321 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4381 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4441 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4501 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa
     4561 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     4621 aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     4681 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc
     4741 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     4801 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     4861 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat
     4921 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     4981 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     5041 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     5101 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     5161 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     5221 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     5281 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     5341 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     5401 tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa
     5461 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     5521 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     5581 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     5641 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     5701 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     5761 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     5821 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     5881 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5941 ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
