LOCUS       sequence-205276-        5762 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses PseH from C. jejuni with an N-terminal 2xHis Gln 5x His 
            feature, followed by the TEV cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5762)
  AUTHORS   Zamora CY, Schocker NS, Chang MM, Imperiali B
  TITLE     Chemoenzymatic Synthesis and Applications of Prokaryote-Specific 
            UDP-Sugars.
  JOURNAL   Methods Enzymol. 2017;597:145-186. doi: 10.1016/bs.mie.2017.06.003. 
            Epub 2017 Jul 5.
  PUBMED    28935101
REFERENCE   2  (bases 1 to 5762)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods 
            Enzymol."; date: "2017"; volume: "597:145-186. doi"; pages: " 
            10.1016/bs.mie.2017.06.003. Epub 2017 Jul 5"
COMMENT     Sequence Label: His8-TEV-PseH
FEATURES             Location/Qualifiers
     source          1..5762
                     /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             116..136
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             623..640
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(693..711)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      707..754
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      791..1246
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(878..897)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1088..1109
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1338..2153)
                     /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(1338..2153)
                     /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     2061..2080
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2145..2164
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2275..2863
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2764..2783
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3017..3034
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3049..3188
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3114..3133)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3274..3296
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3290..3481)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4290..5372)
                     /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     5334..5353
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5373..5450)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5656..5675
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcag catcatcatc atcacgaaaa
      121 tctgtatttt cagagcggat ccatgataaa acttaaaaat ttcgcagaac ttaattctca
      181 agaaataaag ctgattttta aatggcgcaa tcaccccgat atcagtcaat ttatgaaaac
      241 aaaacatatt gattttgaag aacatttaag atttataaga aacttacatc aagattctaa
      301 taaaaaatat tttttagttt ttcaagatga acagattatc ggtgtaattg attttgtcaa
      361 tatcacaact aaatcttgcg aatttggact ttatgcaata cctgatttaa aaggcgtagg
      421 acaagtttta atgaatgaaa tcaaaaaata cgcctttgaa attttaaaag tagatactct
      481 taaagcttat gtttttaaag acaatcacaa agccttaaaa ttatatcaac aaaatcattt
      541 tactatttat gatgaagata aagatttcta ctatgtttgc ttaaagcaat ctcactgcaa
      601 agccttgcct agctaactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
      661 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
      721 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
      781 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
      841 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
      901 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      961 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1021 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1081 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     1141 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1201 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     1261 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1321 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     1381 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     1441 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     1501 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     1561 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     1621 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     1681 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     1741 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     1801 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     1861 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     1921 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     1981 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     2041 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     2101 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     2161 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     2221 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2281 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2341 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2401 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2461 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2521 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2581 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2641 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2701 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2761 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2821 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2881 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2941 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3001 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3061 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3121 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3181 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3241 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3301 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     3361 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     3421 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     3481 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     3541 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     3601 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     3661 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     3721 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     3781 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     3841 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     3901 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     3961 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4021 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4081 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4141 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4201 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4261 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4321 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4381 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     4441 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4501 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4561 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     4621 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     4681 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     4741 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     4801 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     4861 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     4921 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     4981 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5041 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5101 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5161 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5221 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5281 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     5341 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     5401 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     5461 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     5521 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     5581 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     5641 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     5701 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa
     5761 at
//
