LOCUS       sequence-359678-        6040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET30 vector with sfGFP-S-tag inserted between the Bgl II and T7 
            terminator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6040)
  AUTHORS   Jew K, Smith PEJ, So B, Kasman J, Oza JP, Black MW
  TITLE     Characterizing and Improving pET Vectors for Cell-free Expression.
  JOURNAL   Front Bioeng Biotechnol. 2022 Jun 23;10:895069. doi: 
            10.3389/fbioe.2022.895069. eCollection 2022.
  PUBMED    35814024
REFERENCE   2  (bases 1 to 6040)
  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: "Front 
            Bioeng Biotechnol."; date: "2022-06-23"; volume: "10:895069. doi"; 
            pages: " 10.3389/fbioe.2022.895069. eCollection 2022"
COMMENT     Sequence Label: pET30-T7-lacO-His-sfGFP
FEATURES             Location/Qualifiers
     source          1..6040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     966..985
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1005..1082)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1288..1307
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             1453..1475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1486..1503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1513..1530
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1537..1581
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2239..2286
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             2287..2310
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2365..2383)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2379..2426
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2463..2918
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2550..2569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2760..2781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3010..3825)
                     /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(3010..3825)
                     /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     3733..3752
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3817..3836
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3947..4535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4436..4455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4689..4706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4721..4860
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4786..4805)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4946..4968
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4962..5153)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5962..6040,1..1004))
                     /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"
ORIGIN
        1 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
       61 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
      121 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
      181 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
      241 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
      301 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
      361 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
      421 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
      481 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
      541 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
      601 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
      661 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
      721 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
      781 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
      841 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
      901 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
      961 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     1021 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     1081 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     1141 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     1201 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     1261 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     1321 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttaagaa ggagatatac atatgcacca tcatcatcat
     1501 cattcttctg gtctggtgcc acgcggttct ggtatgaaag aaaccgctgc tgctaaattc
     1561 gaacgccagc acatggacag cccagatctc atgagcaaag gtgaagaact gtttaccggc
     1621 gttgtgccga ttctggtgga actggatggc gatgtgaacg gtcacaaatt cagcgtgcgt
     1681 ggtgaaggtg aaggcgatgc cacgattggc aaactgacgc tgaaatttat ctgcaccacc
     1741 ggcaaactgc cggtgccgtg gccgacgctg gtgaccaccc tgacctatgg cgttcagtgt
     1801 tttagtcgct atccggatca catgaaacgt cacgatttct ttaaatctgc aatgccggaa
     1861 ggctatgtgc aggaacgtac gattagcttt aaagatgatt gcaaatataa aacgcgcgcc
     1921 gttgtgaaat ttgaaggcga taccctggtg aaccgcattg aactgaaagg cacggatttt
     1981 aaagaagatg gcaatatcct gggccataaa ctggaataca actttaatag ccataatgtt
     2041 tatattacgg cggataaaca gaaaaatggc atcaaagcga attttaccgt tcgccataac
     2101 gttgaagatg gcagtgtgca gctggcagat cattatcagc agaatacccc gattggtgat
     2161 ggtccggtgc tgctgccgga taatcattat ctgagcacgc agaccgttct gtctaaagat
     2221 ccgaacgaaa aaggcacgcg ggaccacatg gttctgcacg aatatgtgaa tgcggcaggt
     2281 attacgtgga gccatccgca gttcgaaaaa taagtcgacc ggctgctaac aaagcccgaa
     2341 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2401 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     2461 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2881 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     2941 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3001 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     3061 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     3121 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     3181 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     3241 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     3301 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     3361 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     3421 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     3481 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     3541 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     3601 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     3661 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     3721 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     3781 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     3841 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     3901 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3961 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4021 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4081 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4141 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4201 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4261 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4321 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4381 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4441 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4501 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4561 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4621 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4681 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4741 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     4801 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     4861 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4921 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4981 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     5041 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     5101 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     5161 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     5221 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     5281 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     5341 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     5401 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     5461 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     5521 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     5581 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     5641 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     5701 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     5761 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     5821 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     5881 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5941 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6001 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
//
