LOCUS       sequence-322579-        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-WT-charge+
FEATURES             Location/Qualifiers
     source          1..5795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(94..99,100..116,117..123)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    131..147
                     /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     136..158
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    164..233
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             284..310
                     /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             326..343
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             347..379
                     /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(383..391,392..406)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1880..1897)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2050..2136
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2228..2255
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2274..2365
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2366..2434,2435..3226)
                     /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(2584..2603)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3397..3985
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3886..3905
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4139..4156
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4171..4311
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4237..4256)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4397..4419
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4497..4574
                     /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             4575..5657
                     /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(4594..4613)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
       61 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata
      121 atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagcgccgc tgagaaaaag
      181 cgaagcggca ctgctcttta acaatttatc agacaatctg tgtgggcact cgaccggaat
      241 tatcgattaa ctttattatt aaaaattaaa gaggtatata ttaatgtatc gattaaataa
      301 ggaggaataa accatggggg gttctcatca tcatcatcat catggtatgg ctagcatgac
      361 tggtggacag caaatgggtc gggatctgta cgacgatgac gataaggatc gatggggatc
      421 catggcaaat aaagcggtaa atgactttat tcttgcaatg aattacgata agaagaaact
      481 tctgacccat cagggcgaat cgattgaaaa tcgtttcatc aaagagggta atcagcttcc
      541 cgatgagttt gtagtcatcg aacgtaagaa gcgtagtctc tcgactaata ccagtgatat
      601 ttctgtgacg gccactaacg acagccgcct gtatccggga gccctgttgg tagtggatga
      661 gaccttgctg gagaataatc ctacgttact tgccgtagat cgtgccccga tgacgtattc
      721 aattgatctg ccaggtttgg cttcatccga ttcgttctta caagtggaag acccgagcaa
      781 ttcttctgta cgcggtgcgg taaacgatct gctggcgaag tggcatcaag attatggtca
      841 ggttaataat gttccagccc gcatgcagta tgaaaagatc acggctcaca gtatggaaca
      901 actgaaggtg aagtttggat cagactttga aaagaccgga aattcgctgg atattgattt
      961 taactcagtg cattccggag agaagcagat tcagattgtt aatttcaagc agatttatta
     1021 taccgtgagc gttgacgcag tgaagaatcc gggtgatgtg tttcaagata cagtcactgt
     1081 tgaggattta aaacagcgtg gtatttccgc agagcgtcct ctcgtttata ttagcagtgt
     1141 ggcgtatggt cgccaagtat atttgaagtt agaaacaacg tccaagtctg atgaagtaga
     1201 ggcggcattt gaagcgctca tcaaaggggt caaggtggcc ccacagaccg aggcgaagca
     1261 gattgcggac aatacggaag tgaaggcggt tattctgggt ggcgaccctt cgtccggggc
     1321 ccgcgtcgta acaggcaagg tggatatggt ggaagacttg attcaggaag gatcccgctt
     1381 taccgcggat catcctggtc ttcccatttc gtatactacc agttttcttc gtgacaatgt
     1441 ggtggccacc tttcaaaact ctaccgacta tgtggagacg aaggtcaccg cctaccgtaa
     1501 cggtgatctc cttctggatc attcgggcgc atatgtagcg caatattata ttacttggga
     1561 tgaactgagt tatgatcatc aaggcaagga agtacttact cccaaggcat gggaccgtaa
     1621 tggccaggat cttacggcgc actttactac cagcattcct ctcaaaggta atgtgcgtaa
     1681 tcttagtgtt aaaattcgtg agtgtaccgg acttgcctgg gaatggtggc gtaccgttta
     1741 tgagaaaacc gatctcccac tggtccgtaa gcgtacaatt agtatttggg gaactacact
     1801 gtatcctcag gtagaggata aggttgaaaa tgactaattc gaagcttggc tgttttggcg
     1861 gatgagagaa gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa
     1921 aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag
     1981 aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact
     2041 gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     2101 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     2161 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa
     2221 attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt
     2281 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2341 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2401 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2461 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2521 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2581 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     2641 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2701 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2761 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2821 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2881 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     2941 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3001 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3061 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3121 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3181 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3241 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3301 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3361 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3421 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3481 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3541 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3601 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3661 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3721 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3781 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3841 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3901 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3961 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4021 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4081 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4141 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     4201 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     4261 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4321 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4381 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4441 cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca
     4501 ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     4561 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     4621 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     4681 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     4741 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     4801 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4861 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4921 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4981 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     5041 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     5101 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     5161 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     5221 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     5281 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     5341 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     5401 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     5461 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     5521 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     5581 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5641 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt gatctggttt
     5701 gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa
     5761 gctgtggtat ggctgtgcag gtcgtaaatc actgc
//
