LOCUS       sequence-359679-        5924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET30 vector missing lacO and 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 5924)
  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 5924)
  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-sfGFP
FEATURES             Location/Qualifiers
     source          1..5924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      57..512
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..163)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     354..375
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(604..1419)
                     /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(604..1419)
                     /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     1327..1346
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1411..1430
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1541..2129
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2030..2049
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2283..2300
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2315..2454
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2380..2399)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2540..2562
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2556..2747)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3556..4638)
                     /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     4600..4619
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4639..4716)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4922..4941
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5029..5048
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5029..5047
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             5079..5101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5757..5804
                     /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             5805..5828
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(5883..5901)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5897..5924,1..20)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
       61 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
      121 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      181 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      241 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
      301 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
      361 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
      421 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
      481 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
      541 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
      601 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
      661 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
      721 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
      781 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
      841 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
      901 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
      961 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     1021 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     1081 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     1141 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     1201 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     1261 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     1321 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     1381 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     1441 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1501 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1561 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1621 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1681 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1741 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1801 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1861 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     1921 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     1981 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2041 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2101 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2161 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     2221 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     2281 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     2341 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     2401 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     2461 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2521 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2581 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     2641 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     2701 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     2761 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     2821 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     2881 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     2941 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3001 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3061 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3121 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     3181 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     3241 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     3301 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     3361 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     3421 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     3481 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     3541 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3601 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     3661 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     3721 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     3781 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     3841 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     3901 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     3961 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4021 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4081 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4141 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4201 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     4261 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     4321 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     4381 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4441 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     4501 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     4561 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     4621 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4681 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     4741 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     4801 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     4861 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     4921 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     4981 cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta atacgactca
     5041 ctatagggag accacaacgg tttccctcta gaaataattt tgtttaactt taagaaggag
     5101 atatacatat gagcaaaggt gaagaactgt ttaccggcgt tgtgccgatt ctggtggaac
     5161 tggatggcga tgtgaacggt cacaaattca gcgtgcgtgg tgaaggtgaa ggcgatgcca
     5221 cgattggcaa actgacgctg aaatttatct gcaccaccgg caaactgccg gtgccgtggc
     5281 cgacgctggt gaccaccctg acctatggcg ttcagtgttt tagtcgctat ccggatcaca
     5341 tgaaacgtca cgatttcttt aaatctgcaa tgccggaagg ctatgtgcag gaacgtacga
     5401 ttagctttaa agatgatggc aaatataaaa cgcgcgccgt tgtgaaattt gaaggcgata
     5461 ccctggtgaa ccgcattgaa ctgaaaggca cggattttaa agaagatggc aatatcctgg
     5521 gccataaact ggaatacaac tttaatagcc ataatgttta tattacggcg gataaacaga
     5581 aaaatggcat caaagcgaat tttaccgttc gccataacgt tgaagatggc agtgtgcagc
     5641 tggcagatca ttatcagcag aataccccga ttggtgatgg tccggtgctg ctgccggata
     5701 atcattatct gagcacgcag accgttctgt ctaaagatcc gaacgaaaaa ggcacgcggg
     5761 accacatggt tctgcacgaa tatgtgaatg cggcaggtat tacgtggagc catccgcagt
     5821 tcgaaaaata agtcgaccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     5881 caccgctgag caataactag cataacccct tggggcctct aaac
//
