LOCUS       sequence-309117-        6304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Laforin in E.coli cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6304)
  AUTHORS   Choi J, Bong SM, Yang SW, Jang H, Park S, Kim SJ, Lee BI
  TITLE     Recombinant Human Laforin Expressed in Insect Cells: Expression, 
            Purification, and Biochemical Characterizations
  JOURNAL   J Kor Chem Soc, 2015; 59(5): 466-470.
REFERENCE   2  (bases 1 to 6304)
  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: "J Kor Chem 
            Soc,"; date: "2015"; volume: " 59(5)"; pages: " 466-470"
COMMENT     Sequence Label: pHis-Laforin
FEATURES             Location/Qualifiers
     source          1..6304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             87..104
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             126..146
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1149..1172
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1235..1253)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1249..1296
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1333..1788
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1420..1439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1630..1651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1880..2695)
                     /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(1880..2695)
                     /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     2603..2622
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2687..2706
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2817..3405
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3306..3325
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3559..3576
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3591..3730
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3656..3675)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3816..3838
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3832..4023)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4832..5914)
                     /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     5876..5895
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5915..5992)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6198..6217
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gatttaaatt
       61 aactttaaga aggagatata catatgcatc accaccatca ccatggatcg ctggttccga
      121 gatctgaaaa cctgtatttt cagggatcca tgcgcttccg ctttggggtg gtggtgccac
      181 ccgccgtggc cggcgcccgg ccggagctgc tggtggtggg gtcgcggccc gagctggggc
      241 gttgggagcc gcgcggtgcc gtccgcctga ggccggccgg caccgcggcg ggcgacgggg
      301 ccctggccct gcaggagccg ggcctgtggc tcggggaggt ggagctggcg gccgaggagg
      361 cggcgcagga cggggcggag ccgggccgcg tggacacgtt ctggtacaag ttcctgaagc
      421 gggagccggg aggagagctc tcctgggaag gcaatggacc tcatcatgac cgttgctgta
      481 cttacaatga aaacaacttg gtggatggtg tgtattgtct cccaatagga cactggattg
      541 aggccactgg gcacaccaat gaaatgaagc acacaacaga cttctatttt aatattgcag
      601 gccaccaagc catgcattat tcaagaattc taccaaatat ctggctgggt agctgccctc
      661 gtcaggtgga acatgtaacc atcaaactga agcatgaatt ggggattaca gctgtaatga
      721 atttccagac tgaatgggat attgtacaga attcctcagg ctgtaaccgc tacccagagc
      781 ccatgactcc agacactatg attaaactat atagggaaga aggcttggcc tacatctgga
      841 tgccaacacc agatatgagc accgaaggcc gagtacagat gctgccccag gcggtgtgcc
      901 tgctgcatgc gctgctggag aagggacaca tcgtgtacgt gcactgcaac gctggggtgg
      961 gccgctccac cgcggctgtc tgcggctggc tccagtatgt gatgggctgg aatctgagga
     1021 aggtgcagta tttcctcatg gccaagaggc cggctgtcta cattgacgaa gaggccttgg
     1081 cccgggcaca agaagatttt ttccagaaat ttgggaaggt tcgttcttct gtgtgtagcc
     1141 tgctcgagca ccaccaccac caccaccacc actaattgat taatacctag gctgctaaac
     1201 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1261 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1321 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1381 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1441 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1501 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1561 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1621 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1681 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1741 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1801 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1861 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     1921 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1981 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2041 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2101 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2161 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2221 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2281 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2341 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2401 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2461 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2521 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2581 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2641 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2701 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     2761 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2821 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2881 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2941 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3001 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3061 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3121 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3181 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3241 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3301 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3361 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3421 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3481 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3541 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3601 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3661 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3721 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3781 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3841 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3901 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3961 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4021 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4081 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4141 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4201 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4261 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4321 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4381 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4441 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     4501 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4561 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4621 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4681 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4741 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     4801 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4861 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4921 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     4981 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     5041 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5101 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5161 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5221 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5281 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5341 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5401 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5461 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5521 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5581 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5641 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5701 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     5761 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     5821 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     5881 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     5941 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6001 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6061 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6121 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6181 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6241 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     6301 aaat
//
