LOCUS       sequence-322573-        5795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of inserted PLY variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5795)
  AUTHORS   Shah NR, Voisin TB, Parsons ES, Boyd CM, Hoogenboom BW, Bubeck D
  TITLE     Structural basis for tuning activity and membrane specificity of 
            bacterial cytolysins.
  JOURNAL   Nat Commun. 2020 Nov 16;11(1):5818. doi: 10.1038/s41467-020-19482-6.
  PUBMED    33199689
REFERENCE   2  (bases 1 to 5795)
  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: "2020-11-16"; volume: "11(1):5818. doi"; pages: " 
            10.1038/s41467-020-19482-6"
COMMENT     Sequence Label: pTricHis-PLY
FEATURES             Location/Qualifiers
     source          1..5795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(84..89,90..106,107..113)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    121..137
                     /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)."
     primer_bind     126..148
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    154..223
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             274..300
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             316..333
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             337..369
                     /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             join(373..381,382..396)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1870..1887)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2040..2126
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2218..2245
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2264..2355
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2356..2424,2425..3216)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2574..2593)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3387..3975
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3876..3895
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4129..4146
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4161..4301
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4227..4246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4387..4409
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4487..4564
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4565..5647
                     /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     complement(4584..4603)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct
       61 ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa
      121 ttgtgagcgg ataacaattt cacacaggaa acagcgccgc tgagaaaaag cgaagcggca
      181 ctgctcttta acaatttatc agacaatctg tgtgggcact cgaccggaat tatcgattaa
      241 ctttattatt aaaaattaaa gaggtatata ttaatgtatc gattaaataa ggaggaataa
      301 accatggggg gttctcatca tcatcatcat catggtatgg ctagcatgac tggtggacag
      361 caaatgggtc gggatctgta cgacgatgac gataaggatc gatggggatc catggcaaat
      421 aaagcggtaa atgactttat tcttgcaatg aattacgata agaagaaact tctgacccat
      481 cagggcgaat cgattgaaaa tcgtttcatc aaagagggta atcagcttcc cgatgagttt
      541 gtagtcatcg aacgtaagaa gcgtagtctc tcgactaata ccagtgatat ttctgtgacg
      601 gccactaacg acagccgcct gtatccggga gccctgttgg tagtggatga gaccttgctg
      661 gagaataatc ctacgttact tgccgtagat cgtgccccga tgacgtattc aattgatctg
      721 ccaggtttgg cttcatccga ttcgttctta caagtggaag acccgagcaa ttcttctgta
      781 cgcggtgcgg taaacgatct gctggcgaag tggcatcaag attatggtca ggttaataat
      841 gttccagccc gcatgcagta tgaaaagatc acggctcaca gtatggaaca actgaaggtg
      901 aagtttggat cagactttga aaagaccgga aattcgctgg atattgattt taactcagtg
      961 cattccggag agaagcagat tcagattgtt aatttcaagc agatttatta taccgtgagc
     1021 gttgacgcag tgaagaatcc gggtgatgtg tttcaagata cagtcactgt tgaggattta
     1081 aaacagcgtg gtatttccgc agagcgtcct ctcgtttata ttagcagtgt ggcgtatggt
     1141 cgccaagtat atttgaagtt agaaacaacg tccaagtctg atgaagtaga ggcggcattt
     1201 gaagcgctca tcaaaggggt caaggtggcc ccacagaccg agtggaagca gattttggac
     1261 aatacggaag tgaaggcggt tattctgggt ggcgaccctt cgtccggggc ccgcgtcgta
     1321 acaggcaagg tggatatggt ggaagacttg attcaggaag gatcccgctt taccgcggat
     1381 catcctggtc ttcccatttc gtatactacc agttttcttc gtgacaatgt ggtggccacc
     1441 tttcaaaact ctaccgacta tgtggagacg aaggtcaccg cctaccgtaa cggtgatctc
     1501 cttctggatc attcgggcgc atatgtagcg caatattata ttacttggga tgaactgagt
     1561 tatgatcatc aaggcaagga agtacttact cccaaggcat gggaccgtaa tggccaggat
     1621 cttacggcgc actttactac cagcattcct ctcaaaggta atgtgcgtaa tcttagtgtt
     1681 aaaattcgtg agtgtaccgg acttgcctgg gaatggtggc gtaccgttta tgagaaaacc
     1741 gatctcccac tggtccgtaa gcgtacaatt agtatttggg gaactacact gtatcctcag
     1801 gtagaggata aggttgaaaa tgactaattc gaagcttggc tgttttggcg gatgagagaa
     1861 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
     1921 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg
     1981 ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc
     2041 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     2101 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     2161 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga
     2221 aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa
     2281 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2341 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2401 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2461 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2521 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2581 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     2641 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2701 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2761 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2821 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2881 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2941 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3001 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3061 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3121 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3181 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3241 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3301 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3361 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3421 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3481 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3541 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3601 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3661 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3721 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3781 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3841 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3901 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3961 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4021 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4081 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4141 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4201 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4261 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     4321 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4381 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4441 agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg
     4501 gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     4561 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     4621 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     4681 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     4741 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     4801 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     4861 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     4921 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     4981 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     5041 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     5101 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     5161 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     5221 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     5281 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     5341 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     5401 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     5461 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     5521 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     5581 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     5641 gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt gatctggttt gacagcttat
     5701 catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
     5761 ggctgtgcag gtcgtaaatc actgcataat tcgtg
//
