LOCUS       sequence-211446-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses t-4-hydroxy-L-proline dehydratase activating enzyme in E. 
            coli BL21(DE3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  AUTHORS   Levin BJ, Huang YY, Peck SC, Wei Y, Martinez-Del Campo A, Marks JA, 
            Franzosa EA, Huttenhower C, Balskus EP
  TITLE     A prominent glycyl radical enzyme in human gut microbiomes 
            metabolizes trans-4-hydroxy-l-proline.
  JOURNAL   Science. 2017 Feb 10;355(6325). pii: 355/6325/eaai8386. doi: 
            10.1126/science.aai8386.
  PUBMED    28183913
REFERENCE   2  (bases 1 to 7480)
  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: "Science."; 
            date: "2017-02-10"; volume: "355(6325). pii: 355/6325/eaai8386. 
            doi"; pages: " 10.1126/science.aai8386"
COMMENT     Sequence Label: pSV272-PfMBP-HypDAE
FEATURES             Location/Qualifiers
     source          1..7480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        15..33
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    34..58
                     /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             73..95
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             115..132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1342..1362
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2307..2339
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             2346..2363
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2416..2434)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2430..2477
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2514..2969
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2601..2620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2811..2832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3061..3876)
                     /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(3061..3876)
                     /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     3784..3803
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3868..3887
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3998..4586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4487..4506
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4740..4757
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4772..4911
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4837..4856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4997..5019
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5013..5204)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6013..7095)
                     /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"
     promoter        complement(7096..7173)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
       61 tctagaaata attttgttta actttaagaa ggagatatac acatgggttc ttctcaccat
      121 caccatcacc atggtagcgg aagtggaagc ggcagcagcg gcggaaaagt tgttatttgg
      181 catgcaatgc aacccaatga gcttgaggtc ttccaaagct tagcggaaga atacatggca
      241 ctctgtccag aagttgagat agtttttgaa caaaagccaa acttggaaga tgctcttaag
      301 gctgcaatac ccacaggtca aggtcctgac ctctttatct gggctcacga ctggattgga
      361 aagtttgctg aggcaggatt acttgagcca attgatgaat atgtaactga agatctcctt
      421 aacgagtttg ctccaatggc ccaggatgca atgcagtata aaggtcacta ctatgctcta
      481 ccattcgccg ctgaaacagt tgcaataatc tacaacaaag aaatggttag cgagccaccg
      541 aaaacctttg atgagatgaa ggcaataatg gagaagtact atgatccagc aaatgagaag
      601 tatggaatag cttggccaat taatgcctac tttatctcag caattgctca ggcctttggt
      661 ggttactact ttgacgacaa aacagagcaa ccgggactag ataagcctga gacaatcgaa
      721 ggatttaagt tcttcttcac agaaatatgg ccatacatgg ctccaactgg agactacaac
      781 actcaacaga gtatattcct cgagggtaga gccccaatga tggttaatgg tccatggagc
      841 attaacgacg ttaagaaggc aggaataaac tttggagtgg ttccactacc tccaataatc
      901 aaggatggta aggagtactg gccaaggcct tacggtggag ttaagttgat ttacttcgca
      961 gcgggaataa agaacaagga tgctgcatgg aagttcgcaa agtggcttac cacaagcgaa
     1021 gagtcaatta agacattggc actagagctg ggatacatac cggttcttac gaaggttctt
     1081 gatgatccag aaattaagaa tgatccagta atctatggct ttggacaagc agttcagcac
     1141 gcatacctaa tgccaaagag tccaaagatg agtgctgttt ggggcggagt tgatggggca
     1201 attaacgaaa tcctccaaga tccacaaaac gctgacattg aaggaatact aaagaagtat
     1261 caacaagaaa tccttaacaa catgcaagga gggagctcga acaacaacaa caataacaat
     1321 aacaacaacc tcgggatcga ggaaaacctg tattttcagg gcgcccatat gaatccatta
     1381 gttataaact tacaaaaatg tagcatacat gatggaccag ggataagaag tacagtattt
     1441 tttaaagggt gtccacttga atgtgtttgg tgtcataatc cagagagtca aacatataca
     1501 aaacaagttt tgtataatga agaaagatgt tcaaaatgtg aagcatgtat aaatatatgc
     1561 cctcataagg caatttacaa aggtgaaaca aaaatatgct tagaccaaga taagtgtgag
     1621 ttttgtgaaa cttgcttaga ctattgtgta aacaatgcaa gagagattgt tggtcaagag
     1681 tattctgtaa gagatttggt taaagagata gagaaggata gaattttcta tgaagaatca
     1741 ggtggtggag taactctatc aggtggagaa gtaatggctc aagatatgga cttcatttgt
     1801 ggagtaataa acatgtgcaa atcaaaggga atccatgtag caattgatac atgtggctat
     1861 gcaaagtcag aaaattatga aagagttgct aaatgtgcag atttgttttt atatgatatt
     1921 aagcttattg atgaagataa gcacataaaa tttacaggaa aatcaaatga cttaatactg
     1981 aaaaatgtta agattttaag tgaattaggg gctaacataa atataagaat accattaatt
     2041 gtaggagtaa atgtagatga tgaaaattta gaggttaaaa aaatgataga gtttttaaag
     2101 cctttgaata tacaagcagt aagtcttctt ccatatcata atattggtaa acacaagtat
     2161 gataaaattt ataaaaagta tgaaggagaa gaactacaaa gaccatcaga agaaaaatta
     2221 gaggaaataa aaagactgtt tgaagcaagt aatttcaata ctaagattgg agggtaacgg
     2281 ccgcactcga gatcaaacgg gctagccagc cagaactcgc cccggaagac cccgaggatg
     2341 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     2401 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2461 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2521 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2581 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2641 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2701 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2761 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2821 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2881 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2941 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     3001 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     3061 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     3121 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     3181 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     3241 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     3301 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     3361 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     3421 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     3481 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3541 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     3601 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3661 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     3721 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     3781 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     3841 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     3901 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     3961 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4021 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4081 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4141 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4201 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4261 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4321 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4381 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4441 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4501 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4561 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4621 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4681 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4741 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4801 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4861 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4921 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4981 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5041 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     5101 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     5161 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     5221 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     5281 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     5341 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     5401 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5461 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5521 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5581 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5641 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     5701 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     5761 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     5821 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     5881 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5941 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     6001 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6061 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgcaaggg tggtttttct
     6121 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     6181 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     6241 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     6301 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     6361 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6421 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6481 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6541 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6601 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6661 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6721 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6781 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6841 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6901 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6961 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     7021 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     7081 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     7141 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     7201 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     7261 ggaatggtgc atgcgcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     7321 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     7381 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     7441 actctgcgac atcgtataac gttactggtt tcacagatct
//
