LOCUS       sequence-204719-        7428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LR reaction to create expression plasmid. For prokaryotic expression
            of target protein as N-terminal fusion form with Sumo tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7428)
  AUTHORS   Nguyen AN, Song JA, Nguyen MT, Do BH, Kwon GG, Park SS, Yoo J, Jang 
            J, Jin J, Osborn MJ, Jang YJ, Thi Vu TT, Oh HB, Choe H
  TITLE     Prokaryotic soluble expression and purification of bioactive human 
            fibroblast growth factor 21 using maltose-binding protein.
  JOURNAL   Sci Rep. 2017 Nov 23;7(1):16139. doi: 10.1038/s41598-017-16167-x.
  PUBMED    29170489
REFERENCE   2  (bases 1 to 7428)
  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: "Sci Rep."; 
            date: "2017-11-23"; volume: "7(1):16139. doi"; pages: " 
            10.1038/s41598-017-16167-x"
COMMENT     Sequence Label: pDest-Sumo
FEATURES             Location/Qualifiers
     source          1..7428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    46..70
                     /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)."
     RBS             85..107
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             120..143
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             150..443
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     protein_bind    462..586
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(611..616,617..634,635..641)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             695..1354
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(761..780)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1696..2001
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1880..1899
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2042..2166)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2180..2197
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2250..2268)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2264..2311
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2348..2803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2435..2454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2645..2666
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2829..2933
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2934..3002,3003..3794)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3152..3171)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3965..4553
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4454..4473
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4707..4724
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4739..4878
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4804..4823)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4964..4986
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4980..5171)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5980..7062)
                     /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     7024..7043
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7063..7140)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7346..7365
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatccagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
       61 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgaaac
      121 accatcacca tcaccatcac catgagctca tgtcggactc agaagtcaat caagaagcta
      181 agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag gtgtccgatg
      241 gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg ctgatggaag
      301 cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac gacggtatta
      361 gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat attattgagg
      421 ctcacagaga acagattggt gggggtaccg gatcttacat cacaagtttg tacaaaaaag
      481 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
      541 aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg
      601 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga
      661 tccgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
      721 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
      781 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
      841 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
      901 tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
      961 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     1021 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     1081 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     1141 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     1201 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     1261 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     1321 acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct tactaaaagc
     1381 cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat
     1441 gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt
     1501 gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa
     1561 gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc
     1621 aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga
     1681 acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     1741 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc
     1801 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     1861 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
     1921 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     1981 ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga
     2041 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     2101 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa
     2161 agtggtgatg tacctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     2221 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     2281 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     2341 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2401 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2461 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2521 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2581 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2641 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2701 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2761 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     2821 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2881 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2941 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3001 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3061 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3121 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3181 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3241 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3301 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3361 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3421 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     3481 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3541 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3601 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3661 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3721 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3781 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3841 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3901 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3961 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4021 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4081 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     4141 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4201 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4261 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4321 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4381 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4441 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4501 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4561 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4621 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4681 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4741 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     4801 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4861 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4921 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4981 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     5041 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     5101 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     5161 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     5221 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     5281 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     5341 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     5401 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     5461 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     5521 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     5581 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     5641 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     5701 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     5761 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     5821 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     5881 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     5941 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6001 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6061 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     6121 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     6181 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     6241 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     6301 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     6361 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     6421 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     6481 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     6541 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     6601 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     6661 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     6721 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     6781 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     6841 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     6901 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     6961 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     7021 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     7081 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     7141 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     7201 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     7261 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     7321 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     7381 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcg
//
