LOCUS       sequence-368621-        5831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal (His)6-Thrombin cleavage site fusion of human 
            gene or portion of gene with point mutation in bacterial strains. 
            pET based vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5831)
  AUTHORS   Adams BM, Coates MN, Jackson SR, Jurica MS, Davis TL
  TITLE     Nuclear cyclophilins affect spliceosome assembly and function in 
            vitro.
  JOURNAL   Biochem J. 2015 Jul 15;469(2):223-33. doi: 10.1042/BJ20150396. Epub 
            2015 May 13.
  PUBMED    25967372
REFERENCE   2  (bases 1 to 5831)
  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: 
            "10.1042/BJ20150396"; journalName: "Biochem J"; date: "2015-07-15- 
            15"; volume: "469"; issue: "2"; pages: "223-33"
COMMENT     Sequence Label: ppwd1.473.646.W601A
FEATURES             Location/Qualifiers
     source          1..5831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             108..125
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             677..694
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(747..765)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      761..808
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      845..1300
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(932..951)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1142..1163
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1392..2207)
                     /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(1392..2207)
                     /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     2115..2134
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2199..2218
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2329..2917
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2818..2837
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3071..3088
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3103..3242
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3168..3187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3328..3350
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3344..3535)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4344..5426)
                     /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     5388..5407
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5427..5504)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5710..5729
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5813..5831,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5813..5831
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gttccgcgtg
      121 gtagtccttc taaagaagaa gtcatggcag ctactcaagc tgaaggacct aaacgagttt
      181 cggacagtgc cattatccac accagcatgg gagacattca caccaaactt tttcctgttg
      241 agtgccctaa gacagtggaa aacttctgtg ttcacagcag aaatggttat tataatgggc
      301 atacatttca ccgtataatt aagggcttta tgattcagac tggagatcca acaggtactg
      361 gtatgggagg agaaagcata tggggaggag aatttgaaga tgaatttcat tcaacattac
      421 gacatgacag gccgtacaca ctcagcatgg ctaacgcggg atcaaatact aatggatccc
      481 agtttttcat aacggtagta ccaacgcctg cgcttgataa taagcataca gtatttggac
      541 gagtgactaa aggaatggaa gttgtacaga ggatctccaa cgtcaaagtc aatcccaaaa
      601 cagataagcc ctatgaggat gtcagcatca taaatattac tgtcaagtga tgacgaagct
      661 tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
      721 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
      781 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
      841 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
      901 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
      961 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1021 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1081 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1141 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1201 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1261 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     1321 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1381 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     1441 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     1501 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     1561 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     1621 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     1681 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     1741 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     1801 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     1861 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     1921 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     1981 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2041 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2101 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2161 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     2221 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     2281 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2341 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2401 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2461 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2521 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2581 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2641 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     2701 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2761 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2821 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     2881 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2941 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3001 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3061 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3121 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     3181 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3241 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3301 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3361 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3421 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3481 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3541 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3601 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     3661 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     3721 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     3781 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     3841 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     3901 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     3961 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4021 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4081 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4141 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4201 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4261 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4321 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4381 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4441 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4501 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4561 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4621 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4681 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4741 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4801 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4861 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4921 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4981 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5041 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5101 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5161 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5221 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5281 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5341 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5401 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5461 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5521 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5581 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     5641 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     5701 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     5761 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
     5821 ctcactatag g
//
