LOCUS       sequence-207591-        7484 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 7484)
  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 7484)
  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..7484
                     /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(3062..3877)
                     /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(3062..3877)
                     /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     3785..3804
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3869..3888
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3999..4587
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4488..4507
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4741..4758
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4773..4915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4841..4860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5001..5023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5017..5208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6017..7099)
                     /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(7100..7177)
                     /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 atattaacgt ttacaatttc aggtggcact tttcggggaa atgtgcgcgg
     3001 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     3061 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     3121 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     3181 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3241 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     3301 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     3361 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     3421 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     3481 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3541 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     3601 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3661 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3721 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     3781 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3841 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     3901 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3961 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4021 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4081 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4141 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4201 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4261 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4321 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4381 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4441 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4501 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4561 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4621 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4681 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4741 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4801 ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg
     4861 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4921 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4981 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5041 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     5101 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     5161 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     5221 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     5281 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     5341 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     5401 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     5461 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     5521 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     5581 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     5641 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     5701 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     5761 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     5821 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     5881 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     5941 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     6001 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6061 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgca agggtggttt
     6121 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     6181 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     6241 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     6301 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     6361 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     6421 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     6481 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6541 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6601 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6661 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6721 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6781 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6841 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6901 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6961 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     7021 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     7081 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     7141 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     7201 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     7261 gcaaggaatg gtgcatgcgc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     7321 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     7381 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     7441 gcatactctg cgacatcgta taacgttact ggtttcacag atct
//
