LOCUS       sequence-318923-        7025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli iPGM containing a C-terminal biotinylation site 
            and His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7025)
  AUTHORS   Wiedmann M, Dranchak PK, Aitha M, Queme B, Collmus CD, Kashipathy 
            MM, Kanter L, Lamy L, Rogers JM, Tao D, Battaile KP, Rai G, Lovell 
            S, Suga H, Inglese J
  TITLE     Structure-activity relationship of ipglycermide binding to 
            phosphoglycerate mutases.
  JOURNAL   J Biol Chem. 2021 Apr 1:100628. doi: 10.1016/j.jbc.2021.100628.
  PUBMED    33812994
REFERENCE   2  (bases 1 to 7025)
  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: "J Biol 
            Chem. 2021 Apr 1:100628. doi: 10.1016/j.jbc.2021.100628."
COMMENT     Sequence Label: pET21a Ec-iPGM-bio
FEATURES             Location/Qualifiers
     source          1..7025
                     /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             89..121
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1679..1723
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1724..1741
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1757..1774
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1827..1845)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1841..1888
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1925..2380
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2012..2031)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2222..2243
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2406..2510
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2511..2579,2580..3371)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2729..2748)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3542..4130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4031..4050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4284..4301
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4316..4455
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4381..4400)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4541..4563
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4557..4748)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5557..6639)
                     /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     6601..6620
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6640..6717)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6923..6942
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggctagcatg actggtggac agcaaatggg
      121 tcgcggatcc atgttggttt ctaaaaaacc tatggtactg gtgattctgg atggctatgg
      181 ctatcgtgaa gaacagcagg ataacgccat ttttagtgct aaaaccccgg taatggatgc
      241 actgtgggcc aatcgtccgc ataccctgat cgacgcttcc ggtctggaag tcggtctgcc
      301 tgaccgtcag atgggtaact ccgaagtagg ccacgttaac ctgggtgccg gccgcatcgt
      361 gtatcaggac ctgactcgtc tggacgttga aatcaaagat cgcgctttct ttgctaatcc
      421 agtgctgact ggcgcggtag ataaagcgaa aaacgcaggt aaggcagtac acattatggg
      481 tctgctctcc gcaggcggcg tacacagcca cgaagatcac atcatggcga tggtagaact
      541 ggcagctgaa cgcggcgcag aaaaaatcta cctgcacgca ttccttgacg gtcgcgacac
      601 cccgccgcgc agtgctgaat cctcgctgaa aaaatttgaa gagaaatttg ctgcgctggg
      661 taaaggccgc gtagcgtcca tcattggtcg ttactacgcg atggaccgcg ataaccgttg
      721 ggatcgcgtt gaaaaagcct atgacctgct gactctggcg cagggtgagt tccaggccga
      781 tactgccgtt gctggtttgc aggcagctta tgctcgcgac gaaaacgacg agttcgtgaa
      841 agcgaccgtt attcgtgcag aaggtcagcc agatgcggca atggaagacg gcgatgcgct
      901 gattttcatg aacttccgtg ctgaccgcgc gcgcgaaatc actcgtgcat tcgtaaacgc
      961 agatttcgac ggcttcgcgc gtaagaaagt ggttaacgtc gatttcgtga tgctgaccga
     1021 atacgccgct gacatcaaaa ctgcggttgc ttacccaccc gcttccctgg ttaacacctt
     1081 cggcgagtgg atggcgaaaa acgacaaaac tcagttgcgt atttccgaaa ccgaaaaata
     1141 tgcccacgtt actttcttct tcaacggtgg cgtagaagag tcgttcaaag gcgaagatcg
     1201 cattctgatc aactcaccga aagtggctac ctacgatctg caaccggaaa tgagctccgc
     1261 agagctgacc gaaaaactgg ttgcggcaat caagagcggc aaatacgaca ccattatctg
     1321 taactatccg aacggcgaca tggtaggtca caccggggtg atggaagcgg cggttaaagc
     1381 agttgaagcg ctggatcact gcgtggaaga agtcgcgaaa gcggttgagt ccgtgggtgg
     1441 acaactgctg atcaccgctg accacggtaa cgctgaacag atgcgcgatc cggcaacggg
     1501 tcaggcacac acggcacaca ccaacctgcc agttccgctg atttacgttg gtgataagaa
     1561 cgtgaaagcg gttgcaggcg gcaaactttc tgacatcgcg ccgaccatgt tgtcgctgat
     1621 gggtatggaa atcccgcaag agatgactgg taagccgctg ttcatcgtgg aactcgaggg
     1681 tctgaacgac atcttcgagg ctcagaaaat cgaatggcac gaattagtgc ctcgcggaag
     1741 cgctgcaggg ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     1801 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1861 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1921 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1981 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2041 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2101 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2161 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2221 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2281 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2341 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     2401 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2461 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2521 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2581 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2641 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2701 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     2761 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2821 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2881 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2941 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3001 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     3061 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3121 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3181 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3241 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3301 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3361 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3421 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3481 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3541 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3601 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3661 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3721 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3781 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3841 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3901 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3961 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4021 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4081 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4141 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4201 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4261 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4321 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     4381 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4441 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4501 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4561 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     4621 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     4681 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4741 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4801 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4861 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4921 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4981 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5041 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5101 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5161 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     5221 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     5281 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     5341 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     5401 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     5461 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     5521 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5581 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5641 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5701 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5761 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5821 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5881 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5941 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6001 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6061 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     6121 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     6181 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     6241 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     6301 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     6361 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     6421 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     6481 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     6541 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6601 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6661 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6721 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6781 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     6841 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     6901 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     6961 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     7021 gaaat
//
