LOCUS       sequence-308627-        6010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET28a with a design flaw in the T7 promoter fixed. Contains the 
            coding sequence for sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6010)
  AUTHORS   Shilling PJ, Mirzadeh K, Cumming AJ, Widesheim M, Kock Z, Daley DO
  TITLE     Improved designs for pET expression plasmids increase protein 
            production yield in Escherichia coli.
  JOURNAL   Commun Biol. 2020 May 7;3(1):214. doi: 10.1038/s42003-020-0939-8.
  PUBMED    32382055
REFERENCE   2  (bases 1 to 6010)
  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: "2020-05-7"; volume: "3(1):214. doi"; pages: " 
            10.1038/s42003-020-0939-8"
COMMENT     Sequence Label: pET28a T7pCONS sfGFP
FEATURES             Location/Qualifiers
     source          1..6010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(84..1166)
                     /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     1128..1147
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1167..1244)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1450..1469
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1553..1572
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1553..1571
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1576..1600
                     /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             1615..1637
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1656..1673
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1683..1700
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(1704..1706,1707..2420)
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1704..2417
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1734..1762)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2357..2377
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2427..2444
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2497..2515)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2511..2558
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2595..3050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2682..2701)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2892..2913
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3142..3957)
                     /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(3142..3957)
                     /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     3865..3884
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3949..3968
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4079..4667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4568..4587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4821..4838
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4853..4992
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4918..4937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5078..5100
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5094..5285)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
       61 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
      121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
      181 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
      241 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
      301 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
      361 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
      421 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
      481 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
      541 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
      601 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
      661 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
      721 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
      781 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
      841 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
      901 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
      961 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     1021 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     1081 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     1141 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     1201 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     1261 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     1321 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     1381 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     1441 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     1501 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
     1561 ctcactatag ggagaggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     1621 taactttaag aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg
     1681 gcctggtgcc gcgcggcagc catatgagca aaggagaaga acttttcact ggagttgtcc
     1741 caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc cgtggagagg
     1801 gtgaaggtga tgctacaaac ggaaaactca cccttaaatt tatttgcact actggaaaac
     1861 tacctgttcc gtggccaaca cttgtcacta ctctgaccta tggtgttcaa tgcttttccc
     1921 gttatccgga tcacatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg
     1981 tacaggaacg cactatatct ttcaaagatg acgggaccta caagacgcgt gctgaagtca
     2041 agtttgaagg tgataccctt gttaatcgta tcgagttaaa aggtattgat tttaaagaag
     2101 atggaaacat tctcggacac aaactggagt acaactttaa ctcacacaat gtatacatca
     2161 cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacgttgaag
     2221 atggttccgt tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg
     2281 tccttttacc agacaaccat tacctgtcga cacaatctgt tctttcgaaa gatcccaacg
     2341 aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc tgctgggatt acacatggca
     2401 tggatgaact ctacaaataa ctcgagcacc accaccacca ccactgagat ccggctgcta
     2461 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2521 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2581 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2641 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2701 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2761 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2821 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2881 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2941 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3001 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     3061 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3121 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     3181 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     3241 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     3301 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     3361 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     3421 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3481 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     3541 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3601 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3661 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3721 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3781 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3841 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3901 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     3961 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     4021 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4081 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4141 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4201 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4261 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4321 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4381 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4441 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4501 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4561 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4621 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4681 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4741 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4801 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4861 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4921 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4981 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5041 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5101 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     5161 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5221 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5281 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5341 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     5401 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     5461 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5521 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5581 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5641 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5701 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     5761 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     5821 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5881 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5941 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     6001 cggaaggagc
//
