LOCUS       sequence-360470-        4652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mNeongreen-stefinA fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4652)
  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 4652)
  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-StefinA
FEATURES             Location/Qualifiers
     source          1..4652
                     /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             1156..1173
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1189..1233
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1271..1289)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1285..1332
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1562..2377)
                     /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(1562..2377)
                     /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     2285..2304
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2378..2469)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2485..3234)
                     /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(2495..2512)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2735..2754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3430..4512)
                     /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     4474..4493
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4513..4590)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4636..4652,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4636..4652,1..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 gcggaatgat tcccggcggt ttatcagagg caaaacccgc gacgccagag
      901 attcaagaaa tcgtcgacaa agttaaaccg caattggaag agaagaccaa tgaaacttat
      961 ggcaagttag aggccgttca atacaagacc caagttgttg ccggcacgaa ttattacatt
     1021 aaggttcgtg cgggcgacaa taagtacatg cacctcaagg tctttaaatc attgcctggt
     1081 cagaatgagg atctcgtttt aacgggctac caagttgaca agaataaaga cgacgaactc
     1141 actggatttg gtacccatca tcatcatcat cattaactcg agtctggtaa agaaaccgct
     1201 gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag
     1261 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1321 aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact gcttccggta
     1381 gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa
     1441 ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa atgtgcgcgg
     1501 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     1561 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     1621 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     1681 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     1741 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     1801 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     1861 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     1921 gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa acaggaatcg
     1981 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     2041 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     2101 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     2161 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     2221 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     2281 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     2341 gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt caatattatt
     2401 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2461 ataaacaaat aggcatgcag cgctcttccg cttcctcgct cactgactcg ctacgctcgg
     2521 tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc ggtaatacgg ttatccacag
     2581 aatcagggga taaagccgga aagaacatgt gagcaaaaag caaagcaccg gaagaagcca
     2641 acgccgcagg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2701 tcaagccaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2761 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2821 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gttggtatct cagttcggtg
     2881 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2941 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3001 gcagcagcca ttggtaactg atttagagga ctttgtcttg aagttatgca cctgttaagg
     3061 ctaaactgaa agaacagatt ttggtgagtg cggtcctcca acccacttac cttggttcaa
     3121 agagttggta gctcagcgaa ccttgagaaa accaccgttg gtagcggtgg tttttcttta
     3181 tttatgagat gatgaatcaa tcggtctatc aagtcaacga acagctattc cgttactcta
     3241 gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa
     3301 gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     3361 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     3421 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3481 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     3541 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     3601 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     3661 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     3721 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     3781 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     3841 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     3901 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     3961 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     4021 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     4081 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     4141 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     4201 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     4261 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     4321 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     4381 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     4441 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     4501 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     4561 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     4621 cctgcattag gaaattaata cgactcacta ta
//
