LOCUS       sequence-227008-        6979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SdrG N2 and N3, B1 and B2 domains with C-terminal ybbr tag for 
            covalent immobilization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6979)
  AUTHORS   Milles LF, Unterauer EM, Nicolaus T, Gaub HE
  TITLE     Calcium stabilizes the strongest protein fold.
  JOURNAL   Nat Commun. 2018 Nov 12;9(1):4764. doi: 10.1038/s41467-018-07145-6.
  PUBMED    30420680
REFERENCE   2  (bases 1 to 6979)
  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: "Nat 
            Commun."; date: "2018-11-12"; volume: "9(1):4764. doi"; pages: " 
            10.1038/s41467-018-07145-6"
COMMENT     Sequence Label: pET28a-SdrG_N2N3-B1-B2-HIS-ybbr
FEATURES             Location/Qualifiers
     source          1..6979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     83..102
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        83..101
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    102..126
                     /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             141..163
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1854..1871
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1878..1901
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     terminator      2010..2057
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2094..2549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2181..2200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2391..2412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2641..3456)
                     /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(2641..3456)
                     /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     3364..3383
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3448..3467
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3578..4166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4067..4086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4320..4337
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4352..4491
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4417..4436)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4577..4599
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4593..4784)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5593..6675)
                     /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     6637..6656
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6676..6753)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6959..6978
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
       61 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
      121 aattcccctg tagaaataat tttgtttaac tttaagaagg agatatacat atgggtaccg
      181 aacagggtag caatgtgaac catctgatta aagttaccga tcagagcatt accgaaggct
      241 atgatgatag tgatggtatc attaaagcac acgatgccga aaacctgatt tatgatgtta
      301 cctttgaggt ggatgataaa gtgaaaagcg gtgataccat gaccgtgaac attgataaaa
      361 acaccgttcc gagcgatctg accgatagct ttgcaattcc gaaaatcaaa gataacagcg
      421 gtgaaattat tgccaccggc acctatgata ataccaacaa acaaatcacc tacaccttca
      481 ccgattacgt ggataaatac gaaaacatca aagcccatct gaaactgacc agctatatcg
      541 ataaaagcaa agtgccgaac aacaacacca aactggatgt tgagtataaa accgcactga
      601 gcagcgtgaa taaaaccatt accgttgaat atcagaaacc gaacgaaaat cgtaccgcaa
      661 atctgcagag catgtttacc aatatcgata ccaaaaatca taccgtcgaa cagaccattt
      721 atatcaatcc gctgcgttat agcgccaaag aaaccaatgt gaatattagc ggcaatggtg
      781 atgaaggcag caccattatt gatgatagca ccatcatcaa agtgtacaaa gtgggcgata
      841 atcagaatct gccggatagc aatcgcatct atgattacag cgaatatgaa gatgtgacca
      901 acgatgatta tgcacagctg ggtaataata acgacgtgaa tatcaacttc ggcaatattg
      961 atagcccgta tattatcaaa gtgatcagca aatacgaccc gaacaaagat gattacacca
     1021 ccattcagca gaccgttacc atgcagacca ccattaatga atataccggt gaatttcgta
     1081 ccgccagcta tgataacaca attgcattta gcaccagcag cggtcagggt cagggtgatc
     1141 tgcctccgga aaaaacctat aaaatcggtg attatgtgtg ggaggatgtg gataaagatg
     1201 gcattcagaa taccaacgat aacgaaaaac cgctgagcaa tgttctggtt accctgacct
     1261 atccggatgg caccagcaaa agcgttcgta ccgatgaaga aggtaaatat cagtttgatg
     1321 gcctgaaaaa tggcctgacc tacaaaatca cctttgaaac accggaaggt tataccccga
     1381 ccctgaaaca tagcggcacc aatccggcac tggatagcga aggtaatagc gtttgggtta
     1441 caattaatgg ccaggatgat atgaccattg atagcggctt ttatcagacc ccgaaatata
     1501 gcctgggtaa ctatgtttgg tatgacacca ataaagacgg tatccagggt gatgatgaaa
     1561 aaggtattag cggtgttaaa gtgacgctga aagatgaaaa cggcaacatt attagtacca
     1621 ccaccaccga tgaaaatggg aaataccaat tcgacaatct gaacagcggt aactatatcg
     1681 tgcattttga taaaccgagc ggtatgaccc agaccacaac cgatagcggt gatgacgatg
     1741 aacaggatgc agatggtgaa gaagttcacg ttaccattac agatcacgat gatttcagca
     1801 tcgataacgg ctattatgat gatgacagcg agctcaagct tcctaggtct aggcaccacc
     1861 accaccacca cggatccctg gaagttctgt tccagggtcc ggactctctg gaattcatcg
     1921 cttctaaact ggcttaactc gagtaagatc cggctgctaa caaagcccga aaggaagctg
     1981 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2041 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2101 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2161 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2221 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2281 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2341 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2401 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2461 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2521 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2581 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     2641 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     2701 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     2761 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     2821 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2881 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     2941 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     3001 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     3061 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3121 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     3181 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3241 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     3301 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     3361 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     3421 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     3481 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     3541 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3601 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3661 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3721 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3781 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3841 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3901 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3961 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4021 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4081 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4141 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4201 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4261 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4321 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4381 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4441 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4501 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4561 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4621 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     4681 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4741 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4801 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4861 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4921 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4981 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5041 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5101 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5161 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5221 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     5281 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     5341 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     5401 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     5461 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5521 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5581 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5641 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5701 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5761 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5821 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5881 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5941 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6001 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6061 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6121 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6181 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6241 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6301 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6361 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6421 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6481 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6541 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     6601 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6661 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6721 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6781 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6841 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6901 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6961 tgatgtcggc gatataggc
//
