LOCUS       sequence-97108-e        7939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  enhance solubility and correct disulfide bond-formation of fused 
            protein expressed in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7939)
  AUTHORS   Song JA, Koo BK, Chong SH, Kwak J, Ryu HB, Nguyen MT, Vu TT, Jeong 
            B, Kim SW, Choe H
  TITLE     Expression and purification of biologically active human FGF2 
            containing the b'a' domains of human PDI in Escherichia coli.
  JOURNAL   Appl Biochem Biotechnol. 2013 May;170(1):67-80. doi: 
            10.1007/s12010-013-0140-3. Epub 2013 Mar 8.
  PUBMED    23471584
REFERENCE   2  (bases 1 to 7939)
  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"; doi: 
            "10.1007/s12010-013-0140-3"; journalName: "Appl Biochem Biotechnol";
            date: "2013-05"; volume: "170"; issue: "1"; pages: "67-80"
COMMENT     Sequence Label: pDEST-hPDIb′a′
FEATURES             Location/Qualifiers
     source          1..7939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    357..481
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(506..511,512..529,530..536)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             590..1249
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(656..675)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1591..1896
                     /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     1775..1794
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1937..2061)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2075..2092
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2145..2163)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2159..2206
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2243..2698
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2330..2349)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2540..2561
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2725..2829
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2830..2898,2899..3690)
                     /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(3048..3067)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3861..4449
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4350..4369
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4603..4620
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4635..4777
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4703..4722)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4863..4885
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4879..5070)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5879..6961)
                     /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     6923..6942
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6962..7039)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7245..7264
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7354..7373
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7354..7372
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7373..7397
                     /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             7412..7434
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7447..7470
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
ORIGIN
        1 tctacgcccc gtggtgtggg cactgtaaac agctggcgcc gatctgggac aaactgggcg
       61 aaacgtataa agatcacgaa aatattgtga tcgcgaaaat ggattctacc gcgaatgaag
      121 tagaagcggt aaaagtacac tcttttccga cgctgaaatt ctttccagcg agcgcggatc
      181 gtactgtcat tgattataat ggcgaacgta ctctggacgg ctttaagaaa tttctggaaa
      241 gcggcggcca ggatggcgcg ggcgatgatg atgacctgga agacctggaa gaagcggagg
      301 aaccagacat ggaggaggat gacgaccaga aagcggtcgg taccggatct tacatcacaa
      361 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
      421 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
      481 ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg
      541 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa
      601 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
      661 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
      721 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
      781 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
      841 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
      901 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
      961 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     1021 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     1081 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     1141 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     1201 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa cgcgtggatc
     1261 cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag
     1321 aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt
     1381 attacagtga cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata
     1441 tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct
     1501 ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct
     1561 cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga
     1621 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
     1681 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
     1741 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
     1801 gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
     1861 aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc
     1921 cagtctgcag gtcgaccata gtgactggat atgttgtgtt ttacagtatt atgtagtctg
     1981 ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc
     2041 agctttcttg tacaaagtgg tgatgtacct cgagcaccac caccaccacc actgagatcc
     2101 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     2161 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     2221 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2281 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2341 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2401 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2461 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2521 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2581 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2641 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca
     2701 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2761 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2821 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2881 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2941 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3001 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3061 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3121 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3181 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3241 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3301 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3361 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3421 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3481 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3541 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3601 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3661 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3721 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3781 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3841 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3901 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3961 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4021 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4081 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4141 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4201 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4261 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4321 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4381 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4441 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4501 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4561 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4621 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4681 cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4741 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4801 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4861 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     4921 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     4981 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5041 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5101 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5161 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5221 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     5281 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     5341 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     5401 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5461 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5521 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     5581 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     5641 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     5701 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     5761 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5821 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5881 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5941 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     6001 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     6061 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     6121 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     6181 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     6241 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     6301 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     6361 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6421 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6481 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6541 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6601 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6661 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6721 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6781 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6841 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6901 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6961 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     7021 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     7081 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     7141 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7201 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     7261 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     7321 aggatcgaga tccagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     7381 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
     7441 atgaaacacc atcaccatca ccatcaccat gagctcattg aatttacaga acaaacggcg
     7501 ccgaaaattt tcggcggtga gattaaaaca catatcctgc tgtttctgcc gaagagcgtt
     7561 tctgattacg atggtaaact gagtaatttt aaaaccgccg cagaatcttt caaaggtaag
     7621 attctgttca ttttcattga tagcgaccac acggacaatc agcgtatcct ggagttcttt
     7681 ggtctgaaga aagaggaatg cccggctgtg cgtctgatta cgctggaaga ggaaatgaca
     7741 aagtacaagc cggagagcga ggaactgact gcagaacgta tcaccgaatt ttgtcatcgt
     7801 ttcctggagg ggaagattaa gccgcatctg atgagccagg aactgccgga ggattgggac
     7861 aaacagccag tgaaagttct ggtggggaag aattttgaag atgtggcctt cgatgagaag
     7921 aagaatgtgt ttgtggagt
//
