LOCUS       sequence-360466-        5759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mNeongreen-Etanercept fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5759)
  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 5759)
  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-Etanercept
FEATURES             Location/Qualifiers
     source          1..5759
                     /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             2263..2280
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2296..2340
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2378..2396)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2392..2439
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2669..3484)
                     /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(2669..3484)
                     /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     3392..3411
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3485..3576)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3592..4341)
                     /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(3602..3619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3842..3861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4537..5619)
                     /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     5581..5600
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5620..5697)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(5743..5759,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5743..5759,1..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 gcggactgcc tgcacaggtt gcatttaccc cgtatgcacc ggaaccgggt
      901 agcacctgtc gtctgcgtga atattatgat cagaccgcac agatgtgttg tagcaaatgt
      961 agtccgggtc agcatgcaaa agttttttgt accaaaacca gcgataccgt ttgtgatagc
     1021 tgtgaagata gcacctatac gcagctgtgg aattgggttc ctgaatgtct gagctgtggt
     1081 agccgttgta gcagcgatca ggttgaaacc caggcatgta cccgtgaaca gaatcgtatt
     1141 tgtacctgcc gtcctggttg gtattgtgca ctgagcaaac aagagggttg tcgtctgtgt
     1201 gcaccgctgc gtaaatgtcg tccaggtttt ggtgttgcac gtccgggtac agaaaccagt
     1261 gatgttgttt gtaaaccgtg cgcaccgggt acatttagca ataccaccag tagcaccgat
     1321 atttgtcgtc cgcatcagat ttgtaatgtt gttgcaattc cgggtaatgc aagcatggat
     1381 gcagtttgta ccagcaccag tccgacacgt agcatggcac cgggtgcagt tcatctgccg
     1441 cagccggtta gcacccgtag ccagcatacc cagccgactc cggaaccgag caccgcaccg
     1501 agtaccagct ttctgctgcc gatgggtccg agtccgcctg ccgaaggtag caccggtgat
     1561 gaaccgaaaa gctgtgataa aacccatacc tgtccgcctt gtccggctcc ggaactgtta
     1621 ggtggtccga gcgtttttct gtttccgcct aaaccgaaag ataccctgat gattagccgt
     1681 acaccggaag ttacctgtgt tgttgttgat gttagccatg aagatccgga agtgaaattt
     1741 aactggtatg ttgatggtgt ggaagtgcat aatgcaaaaa ccaaaccgcg tgaagaacag
     1801 tataattcaa cctatcgtgt tgttagcgtt ctgaccgttc tgcatcagga ttggctgaat
     1861 ggtaaagaat acaaatgcaa ggtgagcaat aaagcactgc cagcaccgat tgaaaaaacc
     1921 attagcaaag caaaaggtca gcctcgtgaa ccgcaggttt ataccctgcc tccgagccgt
     1981 gaagaaatga ccaaaaatca ggttagcctg acctgtctgg tgaaaggttt ttatccgagc
     2041 gatattgcag ttgaatggga aagcaatggt cagccggaaa ataactataa aaccacaccg
     2101 cctgttctgg atagtgatgg tagctttttt ctgtatagca aactgaccgt tgataaaagc
     2161 cgttggcagc agggtaatgt ttttagctgt agcgttatgc atgaagccct gcataatcat
     2221 tatacccaga aaagcctgag cctgagtccg ggtaaaggta cccatcatca tcatcatcat
     2281 taactcgagt ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg
     2341 tctactagcg cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa
     2401 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag
     2461 aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa
     2521 gcggctattt aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag
     2581 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2641 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     2701 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     2761 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     2821 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2881 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     2941 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     3001 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt
     3061 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     3121 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     3181 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3241 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     3301 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     3361 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     3421 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     3481 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3541 gatacatatt tgaatgtatt tagaaaaata aacaaatagg catgcagcgc tcttccgctt
     3601 cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact
     3661 caaaagcggt aatacggtta tccacagaat caggggataa agccggaaag aacatgtgag
     3721 caaaaagcaa agcaccggaa gaagccaacg ccgcaggcgt ttttccatag gctccgcccc
     3781 cctgacgagc atcacaaaaa tcgacgctca agccagaggt ggcgaaaccc gacaggacta
     3841 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3901 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3961 tcacgctgtt ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4021 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4081 ccggtaagac acgacttatc gccactggca gcagccattg gtaactgatt tagaggactt
     4141 tgtcttgaag ttatgcacct gttaaggcta aactgaaaga acagattttg gtgagtgcgg
     4201 tcctccaacc cacttacctt ggttcaaaga gttggtagct cagcgaacct tgagaaaacc
     4261 accgttggta gcggtggttt ttctttattt atgagatgat gaatcaatcg gtctatcaag
     4321 tcaacgaaca gctattccgt tactctagat ttcagtgcaa tttatctctt caaatgtagc
     4381 acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc
     4441 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4501 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4561 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4621 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4681 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4741 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4801 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4861 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4921 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4981 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     5041 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5101 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5161 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5221 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5281 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5341 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5401 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5461 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5521 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5581 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5641 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5701 gatctcgacg ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//
