LOCUS       sequence-170995-        7027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SPN and IFS complex in E. coli cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7027)
  AUTHORS   Yoon JY, An DR, Yoon HJ, Kim HS, Lee SJ, Im HN, Jang JY, Suh SW
  TITLE     High-resolution crystal structure of Streptococcus pyogenes 
            beta-NAD(+) glycohydrolase in complex with its endogenous inhibitor 
            IFS reveals a highly water-rich interface.
  JOURNAL   J Synchrotron Radiat. 2013 Nov;20(Pt 6):962-7. doi: 
            10.1107/S0909049513020803. Epub 2013 Sep 29.
  PUBMED    24121349
REFERENCE   2  (bases 1 to 7027)
  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 
            Synchrotron Radiat."; date: "2013-11"; volume: "20(Pt 6):962-7. 
            doi"; pages: " 10.1107/S0909049513020803. Epub 2013 Sep 29"
COMMENT     Sequence Label: pET28b(+)-SPN+IFS
FEATURES             Location/Qualifiers
     source          1..7027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             1453..1475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1494..1511
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1521..1538
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3286..3303
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3356..3374)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3370..3417
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3454..3909
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3541..3560)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3751..3772
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4001..4816)
                     /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(4001..4816)
                     /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     4724..4743
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4808..4827
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4938..5526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5427..5446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5680..5697
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5712..5851
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5777..5796)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5937..5959
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5953..6144)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6953..7027,1..1008))
                     /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"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttaagaa ggagatatac catgggcagc agccatcatc
     1501 atcatcatca cagcagcggc ctggtgccgc gcggcagcca tatggttagt ggcaaagaaa
     1561 ataaaaaaag cgatgtcaaa tatgaaacga ccaaagttat ggaagctaac gcaacttcct
     1621 ctaaagaaga caatcatgtc atgcacacat tagacggctc aatgagtact gtctgggagg
     1681 aaaattcacc tggtggtggt gttggtgagg tactttccta caagtttgcg tccccgatgc
     1741 atattgggag aattttaatt gttaatggag acacatctag caaggagaat tactacaaga
     1801 aaaatagaat tgcaaaggct gatgttaaat actataacgg gaataaattg gtcctctttc
     1861 aaaaaattga attaggcgac acctacacta aaaaaccgca tcacattgag attgataaaa
     1921 aattagatgt tgatcgtatt gatattgagg taacagaggt ccatcaggga caaaacaagg
     1981 atattttagc cttgtcagag gtcacttttg gcaatatgga acgcgatatt tttgaaaaaa
     2041 agtttaaaga aattaaagat aaatgggtaa cagataaaca agcagatgag tttattgaaa
     2101 ctgccgacaa gtatgctgat aaagctgttc agatgtctgc tgttgcgtca cgtgctgagt
     2161 attaccggat gtacgttagc cgcaaatacc actacaaaaa agagtttgtt gaaaaactaa
     2221 aacaagtcta caaagaaagc ggagcttctc acgttacaag taaaaaagat gtgatgttag
     2281 cttttgatga tgctaaaaga aagtcaacga ttggtcgaca agaaaacggt ctttttgtga
     2341 caagttttgc tgaagatatg gcattgctct ttactgatca aggtaagtta aaatcagctg
     2401 accaaattga aaatataaaa ggtgtcgata gcggaaaata tagcgatggg gtttatcaat
     2461 atgagtacga ttctgaacta acaaaaaaca ttgataagct aggctatatc cgaacagcta
     2521 gcggagatac tcctggagca aattcgctca acattcctgg ttgccaaacg tggtcaggaa
     2581 aacacattga aaattcagaa agtgagttaa ttttcccatc gattagtgtt aaagatctaa
     2641 aatctaaagc tgtcttagca gagattgatg ccaaaggcta ttttgaaatt attgatccta
     2701 ccatcattgc tccaaatggt gaccataaaa aagtaactgg tcgcttcaaa attaagaaaa
     2761 tgcaagatag aatgcaagat aggaagtaac aatatgtata aggtgccaaa gggtttagaa
     2821 cattaccaaa aaatgtttca aaaagaagta actgttaatg atttaaaaaa atacttgata
     2881 ggtagtgaca aagagtatcg tatcactaga agagactcct atatgggaga tatttctgat
     2941 ccagaagtta ttttagagta tggggtttac cctgctttca taaaaggcta tacccaattg
     3001 aaagctaaca tcgaagaagc attattagaa atgtcaaata gcggtcaagc attagacatt
     3061 taccaagcgg ttcaaaccct aaacgctgaa aacatgttat taaattatta cgaaagcttg
     3121 ccattttatt taaaccgtca aagcatacta gctaatataa ccaaagcgtt aaaagatgcg
     3181 catattagag aggctatggc acattacaaa ttaggagaat ttgctcacta tcaagatact
     3241 atgcttgata tggtcgaaag aacaatagaa acattttaac tcgagcacca ccaccaccac
     3301 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     3361 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     3421 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     3481 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3541 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3601 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3661 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3721 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3781 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3841 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     3901 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3961 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     4021 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     4081 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     4141 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     4201 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     4261 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     4321 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     4381 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     4441 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     4501 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     4561 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     4621 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     4681 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     4741 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     4801 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     4861 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4921 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4981 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5041 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     5101 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5161 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5221 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5281 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5341 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5401 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5461 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5521 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5581 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5641 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5701 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5761 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5821 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5881 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5941 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     6001 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     6061 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     6121 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     6181 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     6241 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     6301 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     6361 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     6421 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     6481 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     6541 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     6601 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     6661 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     6721 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     6781 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     6841 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     6901 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     6961 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     7021 gagaggc
//
