LOCUS       sequence-454243-        6339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LS12 protein expression vector for bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6339)
  AUTHORS   Fukunaga K, Teramoto T, Nakashima M, Ohtani T, Katsuki R, Matsuura 
            T, Yokobayashi Y, Kakuta Y
  TITLE     Structural insights into lab-coevolved RNA-RBP pairs and 
            applications of synthetic riboswitches in cell-free system.
  JOURNAL   Nucleic Acids Res. 2025 Mar 20;53(6):gkaf212. doi: 
            10.1093/nar/gkaf212.
  PUBMED    40119732
REFERENCE   2  (bases 1 to 6339)
  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: "Nucleic 
            Acids Res."; date: "2025-03-20"; volume: "53(6):gkaf212. doi"; 
            pages: " 10.1093/nar/gkaf212"
COMMENT     Sequence Label: pET-His-SUMO-LS12
FEATURES             Location/Qualifiers
     source          1..6339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     177..196
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     280..299
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        280..298
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    299..323
                     /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             338..360
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             376..405
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1119..1137)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1133..1180
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1355..1360,1361..1377,1378..1383))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1410..1428)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1496..1600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1601..1669,1670..2461)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1819..1838)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2632..3220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3121..3140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3374..3391
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3406..3545
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3471..3490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3631..3653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3647..3838)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5150..6232)
                     /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     6194..6213
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6233..6310)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
       61 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
      121 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
      181 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
      241 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
      301 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
      361 tataccatgt caaagcacca ccatcaccac catcatcatc accatggtag tctgcaggat
      421 agtgaagtga accaagaggc aaagccggaa gttaagccag aggtcaagcc cgagacacat
      481 attaacttaa aggtatctga cggtagctct gagatttttt tcaagattaa gaaaacgacc
      541 ccgttgcgcc gtctgatgga ggcctttgcc aagcgtcagg ggaaagaaat ggattcgctg
      601 cgttttcttt atgatggcat tcgtattcag gcagatcagg ctcctgaaga tttggacatg
      661 gaggataacg acattattga ggcacaccgc gaacaaattg gtggcgcaac atcttacgtc
      721 agattcgaag ttcccgaaga tatgcaaaat gaagctcttt cccttcttga gaaggtccgg
      781 gagagcggta aggtcaagaa aggtaccaat tccactactc tggcggtgag ccgcgggttg
      841 gcaaagctgg tatatatcgc cgaagatgtg gacccgccgg aaatcgtagc tcatctccca
      901 cttctttgcg aagagaaaaa tgttccatat atctacgtta aatcaaagaa tgatctgggc
      961 cgagccgtgg gccgtgttta tccgggcgcg tcagccgcta taataaatga aggggagctt
     1021 agaaaggagc ttggtagtct cgtggagaag atcaaaggac tgcaaaaata atagggctgc
     1081 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1141 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1201 cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc
     1261 agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga
     1321 ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt
     1381 caaacatgag aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     1441 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     1501 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1561 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1621 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1681 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1741 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1801 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     1861 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1921 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1981 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2041 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2101 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     2161 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2221 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2281 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2341 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2401 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2461 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2521 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2581 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2641 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2701 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2761 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2821 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2881 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2941 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3001 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3061 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3121 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3181 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3241 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3301 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3361 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3421 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3481 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3541 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3601 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3661 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3721 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3781 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3841 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3901 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3961 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4021 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4081 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4141 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4201 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4261 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc
     4321 tgcccgagat gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc
     4381 aagggttggt ttgcgcattc acagttctcc gcaagaattg attggctcca attcttggag
     4441 tggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca
     4501 tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta caatccatgc
     4561 caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat
     4621 gatcgaagtt aggctggtaa gagccgcgag cgatccttga agctgtccct gatggtcgtc
     4681 atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag
     4741 aagaatcata atggggaagg ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc
     4801 cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4861 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4921 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     4981 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     5041 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     5101 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     5161 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5221 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5281 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5341 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5401 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     5461 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5521 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     5581 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5641 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5701 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5761 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5821 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5881 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5941 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     6001 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     6061 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     6121 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6181 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6241 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6301 cgatggtgtc cgggatctcg acgctctccc ttatgcgac
//
