LOCUS       sequence-356304-        6032 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET30 vector missing lacO 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 6032)
  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 6032)
  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-His-sfGFP
FEATURES             Location/Qualifiers
     source          1..6032
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      56..511
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..162)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     353..374
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(603..1418)
                     /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(603..1418)
                     /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     1326..1345
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1410..1429
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1540..2128
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2029..2048
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2282..2299
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2314..2453
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2379..2398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2539..2561
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2555..2746)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3555..4637)
                     /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     4599..4618
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4638..4715)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4921..4940
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5028..5047
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5028..5046
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             5078..5100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5111..5128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5138..5155
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5162..5206
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             5864..5911
                     /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             5912..5935
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(5990..6008)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(6004..6032,1..19)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
       61 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      121 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      181 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
      241 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
      301 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
      361 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
      421 attttgtcga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
      481 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
      541 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
      601 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
      661 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
      721 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
      781 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
      841 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
      901 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
      961 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     1021 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     1081 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     1141 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     1201 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     1261 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     1321 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     1381 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     1441 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     1501 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1561 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1621 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1681 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1741 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1801 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1861 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1921 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1981 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2041 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2101 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2161 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2221 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2281 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2341 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2401 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2461 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2521 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2581 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2641 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     2701 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     2761 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     2821 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     2881 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     2941 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     3001 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     3061 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     3121 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     3181 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     3241 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     3301 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     3361 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     3421 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3481 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3541 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3601 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3661 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     3721 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     3781 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     3841 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     3901 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     3961 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4021 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4081 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4141 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     4201 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     4261 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     4321 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4381 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     4441 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4501 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4561 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4621 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     4681 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     4741 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     4801 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     4861 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     4921 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     4981 gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac
     5041 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga
     5101 tatacatatg caccatcatc atcatcattc ttctggtctg gtgccacgcg gttctggtat
     5161 gaaagaaacc gctgctgcta aattcgaacg ccagcacatg gacagcccag atctcatgag
     5221 caaaggtgaa gaactgttta ccggcgttgt gccgattctg gtggaactgg atggcgatgt
     5281 gaacggtcac aaattcagcg tgcgtggtga aggtgaaggc gatgccacga ttggcaaact
     5341 gacgctgaaa tttatctgca ccaccggcaa actgccggtg ccgtggccga cgctggtgac
     5401 caccctgacc tatggcgttc agtgttttag tcgctatccg gatcacatga aacgtcacga
     5461 tttctttaaa tctgcaatgc cggaaggcta tgtgcaggaa cgtacgatta gctttaaaga
     5521 tgatggcaaa tataaaacgc gcgccgttgt gaaatttgaa ggcgataccc tggtgaaccg
     5581 cattgaactg aaaggcacgg attttaaaga agatggcaat atcctgggcc ataaactgga
     5641 atacaacttt aatagccata atgtttatat tacggcggat aaacagaaaa atggcatcaa
     5701 agcgaatttt accgttcgcc ataacgttga agatggcagt gtgcagctgg cagatcatta
     5761 tcagcagaat accccgattg gtgatggtcc ggtgctgctg ccggataatc attatctgag
     5821 cacgcagacc gttctgtcta aagatccgaa cgaaaaaggc acgcgggacc acatggttct
     5881 gcacgaatat gtgaatgcgg caggtattac gtggagccat ccgcagttcg aaaaataagt
     5941 cgaccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     6001 taactagcat aaccccttgg ggcctctaaa cg
//
