LOCUS       sequence-368615-        5843 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 5843)
  AUTHORS   Davis TL, Walker JR, Campagna-Slater V, Finerty PJ, Paramanathan R, 
            Bernstein G, MacKenzie F, Tempel W, Ouyang H, Lee WH, Eisenmesser 
            EZ, Dhe-Paganon S
  TITLE     Structural and biochemical characterization of the human cyclophilin
            family of peptidyl-prolyl isomerases.
  JOURNAL   PLoS Biol. 2010 Jul 27;8(7):e1000439. doi: 
            10.1371/journal.pbio.1000439.
  PUBMED    20676357
REFERENCE   2  (bases 1 to 5843)
  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: "PLoS 
            Biol."; date: "2010-07-27"; volume: "8(7):e1000439. doi"; pages: " 
            10.1371/journal.pbio.1000439"
COMMENT     Sequence Label: ppil2.280.457.Y389W
FEATURES             Location/Qualifiers
     source          1..5843
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             708..725
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(778..796)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      792..839
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      876..1331
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(963..982)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1173..1194
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1423..2238)
                     /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(1423..2238)
                     /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     2146..2165
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2230..2249
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2360..2948
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2849..2868
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3102..3119
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3134..3273
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3199..3218)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3359..3381
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3375..3566)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4375..5457)
                     /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     5419..5438
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5458..5535)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5741..5760
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg ttccgcgtgg tagtggctac gtgcggctgc acaccaacaa gggcgacctc
      181 aacctggagc tgcactgcga cctgacacca aaaacctgcg aaaacttcat caggctttgc
      241 aagaagcatt attacgatgg caccatcttc cacagatcca tccggaactt tgtgatccaa
      301 gggggcgacc ccacaggcac aggcacgggt ggggagtcat actgggggaa gcccttcaaa
      361 gacgagttcc ggcccaacct ctcgcacacg ggccgcggca tcctcagcat ggccaactcc
      421 gggcccaaca gcaacaggtc tcaattcttc atcacgtttc gctcctgtgc ctggctggac
      481 aagaagcata ccatctttgg acgggttgtt gggggctttg acgtactgac agccatggag
      541 aatgtggaga gtgaccccaa aactgaccgc cctaaggagg agatccgcat tgatgccact
      601 acagtgttcg tggaccccta tgaggaggcc gatgcccaga ttgcgcagga gcggaagaca
      661 cagctcaagg tagccccgtg atgacgaagc ttgcggccgc actcgagcac caccaccacc
      721 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
      781 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      841 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
      901 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      961 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     1021 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     1081 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     1141 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     1201 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     1261 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     1321 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1381 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     1441 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     1501 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     1561 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     1621 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     1681 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     1741 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     1801 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     1861 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     1921 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     1981 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     2041 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     2101 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     2161 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     2221 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     2281 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2341 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2401 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2461 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2521 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2581 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2641 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2701 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2761 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2821 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2881 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2941 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3001 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3061 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3121 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3181 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     3241 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     3301 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3361 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     3421 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     3481 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     3541 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     3601 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     3661 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     3721 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     3781 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     3841 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     3901 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     3961 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     4021 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     4081 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     4141 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     4201 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     4261 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     4321 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     4381 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4441 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     4501 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     4561 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     4621 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     4681 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     4741 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     4801 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     4861 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     4921 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     4981 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     5041 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     5101 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     5161 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     5221 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     5281 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     5341 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     5401 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     5461 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     5521 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     5581 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     5641 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     5701 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     5761 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     5821 tcgagatctc gatcccgcga aat
//
