LOCUS       sequence-360492-        4856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mNeongreen-Erythropoietin fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4856)
  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 4856)
  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-Erythropoietin
FEATURES             Location/Qualifiers
     source          1..4856
                     /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             1360..1377
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1393..1437
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1475..1493)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1489..1536
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1766..2581)
                     /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(1766..2581)
                     /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     2489..2508
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2582..2673)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2689..3438)
                     /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(2699..2716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2939..2958)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3634..4716)
                     /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     4678..4697
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4717..4794)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4840..4856,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4840..4856,1..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 gcggagcacc gcctcgtctg atttgtgata gccgtgttct ggaacgttat
      901 ctgctggaag caaaagaagc agaaaacatt accaccggtt gtgcagaaca ttgtagcctg
      961 aatgaaaata tcaccgttcc ggataccaaa gtgaactttt atgcatggaa acgtatggaa
     1021 gttggtcagc aggcagttga agtttggcaa ggtctggcac tgctgagcga agcagttctg
     1081 cgtggtcagg ccctgctggt taatagcagc cagccgtggg aaccgctgca actgcatgtt
     1141 gataaagcag ttagcggtct gcgtagcctg acaaccctgc tgcgtgcact gggtgcacag
     1201 aaagaagcaa ttagccctcc ggatgcagca agcgcagcac cgctgcgtac cattaccgca
     1261 gatacctttc gtaaactgtt tcgcgtgtat agcaatttcc tgcgtggtaa actgaaactg
     1321 tataccggtg aagcatgtcg taccggtgat cgtggtaccc atcatcatca tcatcattaa
     1381 ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct
     1441 actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc
     1501 cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag
     1561 cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg
     1621 gctatttaac gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg
     1681 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1741 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     1801 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1861 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     1921 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1981 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     2041 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     2101 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa
     2161 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     2221 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     2281 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     2341 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     2401 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     2461 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     2521 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     2581 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2641 acatatttga atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct
     2701 cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa
     2761 aagcggtaat acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa
     2821 aaagcaaagc accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct
     2881 gacgagcatc acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa
     2941 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3001 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3061 cgctgttggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3121 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3181 gtaagacacg acttatcgcc actggcagca gccattggta actgatttag aggactttgt
     3241 cttgaagtta tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc
     3301 tccaacccac ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc
     3361 gttggtagcg gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca
     3421 acgaacagct attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc
     3481 tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac
     3541 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3601 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3661 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3721 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3781 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3841 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3901 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3961 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     4021 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     4081 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4141 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4201 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4261 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4321 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4381 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4441 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4501 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4561 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4621 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4681 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4741 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     4801 ctcgacgctc tcccttatgc gactcctgca ttaggaaatt aatacgactc actata
//
