LOCUS       sequence-360469-        4111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of stefinA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4111)
  AUTHORS   Eastwood TA, Baker K, Streather BR, Allen N, Wang L, Botchway SW, 
            Brown IR, Hiscock JR, Lennon C, Mulvihill DP
  TITLE     High-yield vesicle-packaged recombinant protein production from E. 
            coli.
  JOURNAL   Cell Rep Methods. 2023 Feb 2;3(2):100396. doi: 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27.
  PUBMED    36936078
REFERENCE   2  (bases 1 to 4111)
  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: "Cell Rep 
            Methods."; date: "2023-02-2"; volume: "3(2):100396. doi"; pages: " 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27"
COMMENT     Sequence Label: pRSFDuet-1_StefinA
FEATURES             Location/Qualifiers
     source          1..4111
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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)."
     CDS             615..632
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             648..692
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(730..748)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      744..791
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1021..1836)
                     /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(1021..1836)
                     /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     1744..1763
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1837..1928)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1944..2693)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(1954..1971)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2194..2213)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2889..3971)
                     /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     3933..3952
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3972..4049)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4095..4111,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgggaggaag cggaatgatt cccggcggtt tatcagaggc aaaacccgcg acgccagaga
      361 ttcaagaaat cgtcgacaaa gttaaaccgc aattggaaga gaagaccaat gaaacttatg
      421 gcaagttaga ggccgttcaa tacaagaccc aagttgttgc cggcacgaat tattacatta
      481 aggttcgtgc gggcgacaat aagtacatgc acctcaaggt ctttaaatca ttgcctggtc
      541 agaatgagga tctcgtttta acgggctacc aagttgacaa gaataaagac gacgaactca
      601 ctggatttgg tacccatcat catcatcatc attaactcga gtctggtaaa gaaaccgctg
      661 ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg
      721 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      781 ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag
      841 tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac
      901 cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga
      961 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     1021 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1081 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1141 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     1201 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     1261 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     1321 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1381 cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga
     1441 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1501 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     1561 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     1621 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     1681 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     1741 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     1801 cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg
     1861 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     1921 taaacaaata ggcatgcagc gctcttccgc ttcctcgctc actgactcgc tacgctcggt
     1981 cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt tatccacaga
     2041 atcaggggat aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg aagaagccaa
     2101 cgccgcaggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2161 caagccagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2221 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2281 tcccttcggg aagcgtggcg ctttctcata gctcacgctg ttggtatctc agttcggtgt
     2341 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2401 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2461 cagcagccat tggtaactga tttagaggac tttgtcttga agttatgcac ctgttaaggc
     2521 taaactgaaa gaacagattt tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa
     2581 gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt ttttctttat
     2641 ttatgagatg atgaatcaat cggtctatca agtcaacgaa cagctattcc gttactctag
     2701 atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag
     2761 ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     2821 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     2881 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2941 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     3001 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     3061 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     3121 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     3181 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     3241 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     3301 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     3361 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     3421 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     3481 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     3541 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     3601 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     3661 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     3721 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     3781 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     3841 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     3901 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     3961 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     4021 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     4081 ctgcattagg aaattaatac gactcactat a
//
