LOCUS       sequence-274488-        5817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7-tagged streptavidin with native C-terminus and periplasmic export
            signal OmpA; controlled by PT7 promoter; KanR, pBR322 ori.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5817)
  AUTHORS   Jeschek M, Reuter R, Heinisch T, Trindler C, Klehr J, Panke S, Ward 
            TR
  TITLE     Directed evolution of artificial metalloenzymes for in vivo 
            metathesis.
  JOURNAL   Nature. 2016 Sep 29;537(7622):661-665. doi: 10.1038/nature19114. 
            Epub 2016 Aug 29.
  PUBMED    27571282
REFERENCE   2  (bases 1 to 5817)
  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"; doi: 
            "10.1038/nature19114"; journalName: "Nature"; date: "2016-09-29- 
            29"; volume: "537"; issue: "7622"; pages: "661-665"
COMMENT     Sequence Label: pET30b_OmpAT7Sav
FEATURES             Location/Qualifiers
     source          1..5817
                     /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)"
     sig_peptide     89..151
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="MKKTAIAIAVALAGFATVAQA"
     CDS             152..181
                     /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="ASMTGGQQMG"
     CDS             678..695
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(748..766)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      762..809
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      846..1301
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(933..952)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1143..1164
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1393..2208)
                     /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(1393..2208)
                     /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     2116..2135
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2200..2219
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2330..2918
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2819..2838
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3072..3089
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3104..3243
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3169..3188)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3329..3351
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3345..3536)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4345..5427)
                     /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     5389..5408
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5428..5505)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5711..5730
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaaaaagaca gctatcgcga ttgcagtggc
      121 actggctggt ttcgctaccg tagcgcaggc cgctagcatg actggtggac agcaaatggg
      181 tcgggatcag gccggcatca ccggcacctg gtacaaccag ctcggctcga ccttcatcgt
      241 gaccgcgggc gccgacggcg ccctgaccgg aacctacgag tcggccgtcg gcaacgccga
      301 gagccgctac gtcctgaccg gtcgttacga cagcgccccg gccaccgacg gcagcggcac
      361 cgccctcggt tggacggtgg cctggaagaa taactaccgc aacgcccact ccgcgaccac
      421 gtggagcggc cagtacgtcg gcggcgccga ggcgaggatc aacacccagt ggctgctgac
      481 ctccggcacc accgaggcca acgcctggaa gtccacgctg gtcggccacg acaccttcac
      541 caaggtgaag ccgtccgccg cctccatcga cgcggcgaag aaggccggcg tcaacaacgg
      601 caacccgctc gacgccgttc agcagtaata aggatccgaa ttcgagctcc gtcgacaagc
      661 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
      721 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      781 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
      841 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
      901 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      961 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1021 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1081 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1141 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1201 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1261 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     1321 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1381 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     1441 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     1501 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     1561 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     1621 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     1681 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     1741 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     1801 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     1861 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     1921 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     1981 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     2041 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2101 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     2161 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     2221 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     2281 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2341 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2401 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2461 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2521 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2581 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2641 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2701 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2761 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2821 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2881 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2941 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3001 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3061 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3121 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     3181 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3241 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3301 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3361 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3421 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3481 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3541 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3601 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     3661 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     3721 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     3781 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     3841 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     3901 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     3961 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     4021 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4081 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4141 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4201 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4261 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4321 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4381 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4441 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4501 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4561 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4621 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     4681 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     4741 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4801 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4861 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4921 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4981 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5041 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5101 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5161 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5221 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5281 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5341 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5401 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5461 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5521 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     5581 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     5641 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     5701 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     5761 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaat
//
