LOCUS       sequence-360460-        5267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mNeongreen-uricase fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5267)
  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 5267)
  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-Uricase
FEATURES             Location/Qualifiers
     source          1..5267
                     /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             1771..1788
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1804..1848
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1886..1904)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1900..1947
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2177..2992)
                     /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(2177..2992)
                     /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     2900..2919
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2993..3084)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3100..3849)
                     /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(3110..3127)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3350..3369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4045..5127)
                     /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     5089..5108
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5128..5205)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(5251..5267,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5251..5267,1..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 gcggaatgag cactacactg tcttcatcga cgtacgggaa ggataacgtt
      901 aagtttctca aggttaagaa ggaccctcag aatccgaaga agcaggaagt tatggaagcc
      961 acggttacgt gcttgttaga gggtggattc gataccagtt acactgaggc ggacaactcg
     1021 tcgattgtgc ccacagatac cgtgaagaac actatcctcg ttcttgcgaa aacgaccgag
     1081 atttggccta tcgagcgctt tgccgcgaag cttgccactc attttgtaga gaaatactcc
     1141 catgtcagcg gcgtttctgt gaaaatcgtt caggatcgtt gggtcaagta cgccgtagac
     1201 ggcaagcccc atgatcacag cttcatccac gagggcggag aaaagcgtat tactgacctt
     1261 tactataagc gcagcggcga ttataagttg tccagtgcga tcaaagacct gaccgtgctg
     1321 aagtcaaccg gcagtatgtt ctatgggtat aataaatgtg attttacgac gttacagcca
     1381 accactgatc gtatccttag tacggacgtc gacgcaacat gggtgtggga caacaagaaa
     1441 atcggaagcg tttatgacat cgctaaagcc gcggacaagg gcatctttga taatgtctac
     1501 aatcaggcgc gcgagattac actgacaacg ttcgctctgg aaaactcccc gagcgtccaa
     1561 gctacaatgt ttaatatggc gacgcaaatc ctggagaaag catgttcggt ttactcggtt
     1621 tcgtacgcgt taccgaataa gcattatttc ctgattgact taaaatggaa gggtctcgaa
     1681 aacgataatg aactgtttta cccgtctccg catccgaatg gcctgatcaa gtgtaccgtg
     1741 gtacgtaagg aaaagacgaa gttaggtacc catcatcatc atcatcatta actcgagtct
     1801 ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca
     1861 gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1921 tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc
     1981 acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa
     2041 cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg
     2101 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2161 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2221 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2281 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2341 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2401 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2461 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2521 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat
     2581 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2641 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2701 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2761 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2821 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2881 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2941 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc
     3001 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3061 aatgtattta gaaaaataaa caaataggca tgcagcgctc ttccgcttcc tcgctcactg
     3121 actcgctacg ctcggtcgtt cgactgcggc gagcggtgtc agctcactca aaagcggtaa
     3181 tacggttatc cacagaatca ggggataaag ccggaaagaa catgtgagca aaaagcaaag
     3241 caccggaaga agccaacgcc gcaggcgttt ttccataggc tccgcccccc tgacgagcat
     3301 cacaaaaatc gacgctcaag ccagaggtgg cgaaacccga caggactata aagataccag
     3361 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3421 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgttgg
     3481 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3541 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3601 gacttatcgc cactggcagc agccattggt aactgattta gaggactttg tcttgaagtt
     3661 atgcacctgt taaggctaaa ctgaaagaac agattttggt gagtgcggtc ctccaaccca
     3721 cttaccttgg ttcaaagagt tggtagctca gcgaaccttg agaaaaccac cgttggtagc
     3781 ggtggttttt ctttatttat gagatgatga atcaatcggt ctatcaagtc aacgaacagc
     3841 tattccgtta ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag
     3901 ccccatacga tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag
     3961 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4021 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4081 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4141 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4201 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4261 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4321 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     4381 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     4441 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4501 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4561 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4621 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4681 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4741 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     4801 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     4861 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     4921 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     4981 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5041 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5101 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5161 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5221 ctcccttatg cgactcctgc attaggaaat taatacgact cactata
//
