LOCUS       sequence-360461-        4838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mNeongreen-DARPinOFF7 fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4838)
  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 4838)
  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_mNeongreen-DARPinOFF7
FEATURES             Location/Qualifiers
     source          1..4838
                     /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"
     CDS             124..831
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     CDS             1342..1359
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1375..1419
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1457..1475)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1471..1518
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1748..2563)
                     /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(1748..2563)
                     /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     2471..2490
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2564..2655)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2671..3420)
                     /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(2681..2698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2921..2940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3616..4698)
                     /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     4660..4679
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4699..4776)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4822..4838,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4822..4838,1..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 tggaggaagc
      121 ggaatggtga gcaagggcga ggaggataac atggcctctc tcccagcgac acatgagtta
      181 cacatctttg gctccatcaa cggtgtggac tttgacatgg tgggtcaggg caccggcaat
      241 ccaaatgatg gttatgagga gttaaacctg aagtccacca agggtgacct ccagttctcc
      301 ccctggattc tggtccctca tatcgggtat ggcttccatc agtacctgcc ctaccctgac
      361 gggatgtcgc ctttccaggc cgcgatggta gatggctccg gataccaagt ccatcgcaca
      421 atgcagtttg aagatggtgc ctcccttact gttaactacc gctacaccta cgagggaagc
      481 cacatcaaag gagaggccca ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg
      541 accaactcgc tgaccgctgc ggactggtgc aggtcgaaga agacttaccc caacgacaaa
      601 accatcatca gtacctttaa gtggagttac accactggaa atggcaagcg ctaccggagc
      661 actgcgcgga ccacctacac ctttgccaag ccaatggcgg ctaactatct gaagaaccag
      721 ccgatgtacg tgttccgtaa gacggagctc aagcactcca agaccgagct caacttcaag
      781 gagtggcaaa aggcctttac cgatgtgatg ggcatggacg agctgtacaa ggctagcaga
      841 tctggaggaa gcggaatggg ttcggactta ggccgcaagt tactggaagc tgcacgcgcg
      901 ggtcaagacg acgaggtacg catccttatg gccaatggtg cggacgtcaa cgccgccgat
      961 aataccggaa caactccttt gcacctcgca gcatattccg gccatttaga gattgtagag
     1021 gtactgctta agcacggtgc cgatgtagat gcgtcggacg tattcgggta cacgcctctc
     1081 cacctcgccg cttattgggg ccacttagaa attgtagagg tgctcttgaa gaatggtgcc
     1141 gacgtgaatg ctatggattc agacggcatg acaccgctcc atctggccgc gaaatggggt
     1201 tatctggaga tcgtcgaggt tctcttgaaa catggcgccg acgtcaatgc acaagataag
     1261 ttcggcaaga ctgccttcga catcagcatc gacaacggta atgaggacct ggccgagatc
     1321 cttcaaaagt tgaatggtac ccatcatcat catcatcatt aactcgagtc tggtaaagaa
     1381 accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta
     1441 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1501 gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt
     1561 ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc
     1621 cctgaaccga cgacaagctg acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt
     1681 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     1741 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     1801 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     1861 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     1921 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     1981 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     2041 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     2101 gtgattgcgc ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag
     2161 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     2221 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     2281 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     2341 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     2401 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     2461 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     2521 atcgcggcct agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat
     2581 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     2641 agaaaaataa acaaataggc atgcagcgct cttccgcttc ctcgctcact gactcgctac
     2701 gctcggtcgt tcgactgcgg cgagcggtgt cagctcactc aaaagcggta atacggttat
     2761 ccacagaatc aggggataaa gccggaaaga acatgtgagc aaaaagcaaa gcaccggaag
     2821 aagccaacgc cgcaggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2881 cgacgctcaa gccagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2941 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3001 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgttg gtatctcagt
     3061 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3121 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3181 ccactggcag cagccattgg taactgattt agaggacttt gtcttgaagt tatgcacctg
     3241 ttaaggctaa actgaaagaa cagattttgg tgagtgcggt cctccaaccc acttaccttg
     3301 gttcaaagag ttggtagctc agcgaacctt gagaaaacca ccgttggtag cggtggtttt
     3361 tctttattta tgagatgatg aatcaatcgg tctatcaagt caacgaacag ctattccgtt
     3421 actctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg
     3481 atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg
     3541 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     3601 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3661 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     3721 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     3781 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     3841 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     3901 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     3961 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4021 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4081 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4141 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4201 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4261 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     4321 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     4381 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     4441 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4501 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     4561 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     4621 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     4681 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4741 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     4801 gcgactcctg cattaggaaa ttaatacgac tcactata
//
