LOCUS       sequence-356301-        5932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET30 vector missing N-terminal His tag 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 5932)
  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 5932)
  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-sfGFP
FEATURES             Location/Qualifiers
     source          1..5932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     496..515
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(535..612)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     818..837
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     925..944
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        925..943
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    944..968
                     /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             983..1005
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1661..1708
                     /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             1709..1732
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(1787..1805)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1801..1848
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1885..2340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1972..1991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2182..2203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2432..3247)
                     /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(2432..3247)
                     /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     3155..3174
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3239..3258
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3369..3957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3858..3877
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4111..4128
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4143..4282
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4208..4227)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4368..4390
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4384..4575)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5384..5932,1..534))
                     /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 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
       61 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
      121 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
      181 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
      241 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
      301 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
      361 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
      421 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
      481 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
      541 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
      601 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
      661 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
      721 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
      781 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
      841 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
      901 agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      961 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatgagcaa
     1021 aggtgaagaa ctgtttaccg gcgttgtgcc gattctggtg gaactggatg gcgatgtgaa
     1081 cggtcacaaa ttcagcgtgc gtggtgaagg tgaaggcgat gccacgattg gcaaactgac
     1141 gctgaaattt atctgcacca ccggcaaact gccggtgccg tggccgacgc tggtgaccac
     1201 cctgacctat ggcgttcagt gttttagtcg ctatccggat cacatgaaac gtcacgattt
     1261 ctttaaatct gcaatgccgg aaggctatgt gcaggaacgt acgattagct ttaaagatga
     1321 tggcaaatat aaaacgcgcg ccgttgtgaa atttgaaggc gataccctgg tgaaccgcat
     1381 tgaactgaaa ggcacggatt ttaaagaaga tggcaatatc ctgggccata aactggaata
     1441 caactttaat agccataatg tttatattac ggcggataaa cagaaaaatg gcatcaaagc
     1501 gaattttacc gttcgccata acgttgaaga tggcagtgtg cagctggcag atcattatca
     1561 gcagaatacc ccgattggtg atggtccggt gctgctgccg gataatcatt atctgagcac
     1621 gcagaccgtt ctgtctaaag atccgaacga aaaaggcacg cgggaccaca tggttctgca
     1681 cgaatatgtg aatgcggcag gtattacgtg gagccatccg cagttcgaaa aataagtcga
     1741 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1801 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1861 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1921 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1981 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2041 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2101 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2161 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2221 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2281 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2341 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2401 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2461 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2521 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     2581 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2641 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     2701 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2761 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     2821 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     2881 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     2941 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3001 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3061 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3121 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3181 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3241 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3301 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3361 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3421 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3481 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3541 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3601 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3661 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3721 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3781 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3841 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3901 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3961 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4021 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4081 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4141 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4201 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4261 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4321 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4381 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4441 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4501 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4561 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4621 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4681 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4741 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4801 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4861 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4921 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4981 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5041 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5101 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5161 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5221 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5281 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5341 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5401 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5461 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5521 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5581 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5641 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     5701 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     5761 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     5821 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     5881 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tg
//
