LOCUS       Exported                6470 bp DNA     circular SYN 08-OCT-2021
DEFINITION  Ligation of Pyl2/Bam+Xho into pET24 H6GST/Bam+Xho.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6470)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 2021 from SnapGene Viewer 5.3.1
            https://www.snapgene.com
COMMENT     Vector molecule:  pET24 H6GST/Bam+Xho
               Fragment ends: XhoI and BamHI
               Fragment size: 5936
            Insert molecule:  Pyl2/Bam+Xho
               Fragment ends: BamHI and XhoI
               Fragment size: 534
FEATURES             Location/Qualifiers
     source          1..6470
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..690
                     /label=H6 GST
                     /note="H6 GST"
                     /note="color: #cc0000; direction: RIGHT"
     CDS             13..30
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     CDS             34..684
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /note="color: #cc99b2"
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     642..664
                     /label=5GEX
                     /note="Genewiz universal primer"
                     /note="color: black; sequence: GGGCTGGCAAGCCACGTTTGGTG"
     misc_feature    691..708
                     /label=thrombin cleavage site
                     /note="color: #cc0000; direction: RIGHT"
     CDS             691..708
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /note="color: #cc99b2
                     Cleavage site after base 702"
                     /translation="LVPRGS"
     misc_feature    709..1236
                     /label=Pyl2
                     /note="aa14-188 from full length protein"
                     /note="color: #cc0000; direction: RIGHT"
     CDS             1243..1260
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     primer_bind     complement(1313..1331)
                     /label=T7 terminator
                     /note="also free Genewiz universal primer"
                     /note="color: black; sequence: GCTAGTTATTGCTCAGCGG"
     terminator      1327..1374
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    1328..1374
                     /label=T7 term
                     /note="color: #cc0000; direction: RIGHT"
     misc_feature    complement(1411..1866)
                     /label=f1 origin
                     /note="color: #cc0000; direction: LEFT"
     rep_origin      1411..1866
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     1941..1980
                     /label=kanR-NotI-rev
                     /note="to PCR out kanR as HindIII-NotI fragment"
                     /note="color: black; sequence: 
                     ATTATTGCGGCCGCTCAGAAAAACTCATCGAGCATC"
     CDS             complement(1959..2774)
                     /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"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1961..1980
                     /label=kanR-NotI-rev
                     /note="to PCR out kanR as HindIII-NotI fragment"
                     /note="color: black; sequence: 
                     ATTATTGCGGCCGCTCAGAAAAACTCATCGAGCATC"
     misc_feature    complement(1962..2774)
                     /label=kanR
                     /note="color: #cc0000; direction: LEFT"
     primer_bind     complement(2757..2776)
                     /label=kanR-HindIII-forward
                     /note="to PCR out kanR as HindIII-NotI fragment"
                     /note="color: black; sequence: 
                     AATAATAAGCTTATGAGCCATATTCAACGG"
     rep_origin      2896..3484
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     misc_feature    3670..3812
                     /label=bom
                     /note="basis of mobility region from pBR322"
                     /note="color: #a6acb3"
     primer_bind     3898..3920
                     /label=3GEX
                     /note="Genewiz universal primer"
                     /note="color: black; sequence: CCGGGAGCTGCATGTGTCAGAGG"
     CDS             complement(3914..4105)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="color: #993366"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4573..4622
                     /label=5GEX
                     /note="Genewiz universal primer"
                     /note="color: black; sequence: GGGCTGGCAAGCCACGTTTGGTG"
     CDS             complement(4914..5996)
                     /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)."
                     /note="color: #993366"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     misc_feature    complement(4917..5996)
                     /label=lacI
                     /note="color: #cc0000; direction: LEFT"
     promoter        complement(5997..6074)
                     /gene="lacI"
                     /label=lacI promoter
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     6077..6094
                     /label=ACYC-Duet-UP1
                     /note="color: black; sequence: GGATCTCGACGCTCTCCC"
     primer_bind     6341..6355
                     /label=pET-Upstream
                     /note="To sequence into orf1 of pETDuet and 
                     pH6Trx-bt-BirA-pETDuet vectors"
                     /note="color: black; sequence: ATGCGTCCGGCGTAG"
     promoter        6383..6401
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    6383..6399
                     /label=T7prom
                     /note="T7lacprom"
                     /note="color: #cc0000; direction: RIGHT"
     protein_bind    6402..6426
                     /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)."
                     /note="color: #31849b"
     RBS             6441..6463
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
                     /note="color: #a6acb3"
ORIGIN
        1 atgaaaaaag gtcatcatca tcatcatcat ggttccccta tactaggtta ttggaaaatt
       61 aagggccttg tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa atatgaagag
      121 catttgtatg agcgcgatga aggtgataaa tggcgaaaca aaaagtttga attgggtttg
      181 gagtttccca atcttcctta ttatattgat ggtgatgtta aattaacaca gtctatggcc
      241 atcatacgtt atatagctga caagcacaac atgttgggtg gttgtccaaa agagcgtgca
      301 gagatttcaa tgcttgaagg agcggttttg gatattagat acggtgtttc gagaattgca
      361 tatagtaaag actttgaaac tctcaaagtt gattttctta gcaagctacc tgaaatgctg
      421 aaaatgttcg aagatcgttt atgtcataaa acatatttaa atggtgatca tgtaacccat
      481 cctgacttca tgttgtatga cgctcttgat gttgttttat acatggaccc aatgtgcctg
      541 gatgcgttcc caaaattagt ttgttttaaa aaacgtattg aagctatccc acaaattgat
      601 aagtacttga aatccagcaa gtatatagca tggcctttgc agggctggca agccacgttt
      661 ggtggtggcg accatcctcc aaaatcggat ctggttccgc gtggatccga gcagaaaacc
      721 ctcgaaccgg ttatcaaaac gtaccaccag ttcgaaccag acccaaccac gtgcacttct
      781 ctcataaccc aacgcatcca cgctccggcc tccgtggttt ggcctcttat ccgccgcttc
      841 gacaaccccg aacgctacaa acactttgta aaaaggtgcc gtctcatctc cggtgatggt
      901 gacgtcggaa gcgtcagaga agtgaccgta atctccggcc tcccagcctc aaccagtacc
      961 gagcggcttg agttcgtcga tgacgaccac cgtgttctaa gcttcagggt cgtcggcgga
     1021 gagcaccgac tcaagaacta caaatcagtg acgtcggtca atgagttctt gaatcaagat
     1081 tccggcaagg tttacacggt ggttcttgaa tcttacaccg ttgatattcc cgagggaaac
     1141 acagaggaag acactaaaat gtttgtggac actgtcgtca aactcaacct tcagaaactc
     1201 ggagttgccg ccacatctgc acctatgcat gattagctcg agcaccacca ccaccaccac
     1261 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1321 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1381 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     1441 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1501 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1561 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1621 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1681 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1741 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1801 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     1861 caatttcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1921 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     1981 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     2041 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     2101 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     2161 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     2221 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     2281 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     2341 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     2401 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     2461 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     2521 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     2581 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     2641 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     2701 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     2761 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     2821 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2881 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2941 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3001 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3061 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3121 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3181 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3241 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3301 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3361 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3421 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3481 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3541 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3601 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3661 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3721 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3781 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3841 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3901 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3961 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4021 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4081 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4141 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4201 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4261 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4321 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4381 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4441 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4501 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4561 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4621 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4681 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4741 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4801 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4861 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4921 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4981 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5041 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5101 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5161 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     5221 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5281 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5341 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5401 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5461 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5521 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5581 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5641 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5701 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5761 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5821 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5881 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5941 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6001 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6061 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6121 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6181 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6241 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6301 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6361 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     6421 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
//
