LOCUS       sequence-298513-        5584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of P. aeruginosa PA14, StrepII-HRV3C-IHFbeta.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5584)
  AUTHORS   Santiago-Frangos A, Buyukyoruk M, Wiegand T, Krishna P, Wiedenheft B
  TITLE     Distribution and phasing of sequence motifs that facilitate CRISPR 
            adaptation.
  JOURNAL   Curr Biol. 2021 Aug 23;31(16):3515-3524.e6. doi: 
            10.1016/j.cub.2021.05.068. Epub 2021 Jun 25.
  PUBMED    34174210
REFERENCE   2  (bases 1 to 5584)
  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: 
            "10.1016/j.cub.2021.05.068"; journalName: "Curr Biol"; date: 
            "2021-08-23- 23"; volume: "31"; issue: "16"; pages: "3515-3524.e6"
COMMENT     Sequence Label: pStrepIHFb
FEATURES             Location/Qualifiers
     source          1..5584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     79..98
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     182..201
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        182..200
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    201..225
                     /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             240..262
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             275..298
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             299..322
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             630..647
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(700..718)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      714..761
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      798..1253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(885..904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1095..1116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1345..2160)
                     /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(1345..2160)
                     /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     2068..2087
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2152..2171
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2282..2870
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2771..2790
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3024..3041
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3056..3195
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3121..3140)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3281..3303
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3297..3488)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4297..5379)
                     /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     5341..5360
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5380..5457)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
       61 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
      121 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
      181 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
      241 ttgtttaact ttaagaagga gatataccat ggattggagc cacccgcagt tcgaaaaact
      301 ggaagtgctg tttcagggtc cgatgaccaa gtcggagttg atcgaacgga tcgttaccca
      361 tcaggggcaa ctgtccgcga aggatgtcga gttggcaatc aagaccatgc tggagcaaat
      421 gtcccaagcc ctggcgactg gggaccggat cgagatccgt ggcttcggca gcttttcctt
      481 gcactaccgc gccccgcgcg tcggtcgcaa ccccaagacc ggggagtcgg tacgcctcga
      541 cggcaagttc gtgccgcact tcaagccggg caaggagttg cgggatcggg tcaacgagcc
      601 ggagtgataa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
      661 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
      721 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
      781 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
      841 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
      901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
      961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1021 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1081 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1141 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1201 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     1261 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1321 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     1381 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     1441 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     1501 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     1561 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     1621 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     1681 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     1741 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     1801 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     1861 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     1921 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     1981 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     2041 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     2101 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     2161 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     2221 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2281 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2341 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2401 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     2461 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2521 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2581 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2641 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2701 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2761 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2821 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2881 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     2941 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3001 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3061 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     3121 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3181 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3241 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3301 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3361 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     3421 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     3481 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     3541 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     3601 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     3661 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     3721 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     3781 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     3841 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     3901 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     3961 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4021 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4081 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4141 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4201 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4261 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4381 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4441 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4501 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4561 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4621 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4681 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4741 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4801 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4861 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4921 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4981 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5041 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5101 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5161 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5221 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5281 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5341 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5401 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5461 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     5521 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5581 cccc
//
