LOCUS       sequence-322580-        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-no charge
FEATURES             Location/Qualifiers
     source          1..5795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(209..214,215..231,232..238)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    246..262
                     /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     251..273
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    279..348
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             399..425
                     /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             441..458
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             462..494
                     /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(498..506,507..521)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1995..2012)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2165..2251
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2343..2370
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2389..2480
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2481..2549,2550..3341)
                     /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(2699..2718)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3512..4100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4001..4020
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4254..4271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4286..4426
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4352..4371)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4512..4534
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4612..4689
                     /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             4690..5772
                     /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(4709..4728)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 agcgcgaatt gatctggttt gacagcttat catcgactgc acggtgcacc aatgcttctg
       61 gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat
      121 tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg
      181 gttctggcaa atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg
      241 tggaattgtg agcggataac aatttcacac aggaaacagc gccgctgaga aaaagcgaag
      301 cggcactgct ctttaacaat ttatcagaca atctgtgtgg gcactcgacc ggaattatcg
      361 attaacttta ttattaaaaa ttaaagaggt atatattaat gtatcgatta aataaggagg
      421 aataaaccat ggggggttct catcatcatc atcatcatgg tatggctagc atgactggtg
      481 gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatcgatgg ggatccatgg
      541 caaataaagc ggtaaatgac tttattcttg caatgaatta cgataagaag aaacttctga
      601 cccatcaggg cgaatcgatt gaaaatcgtt tcatcaaaga gggtaatcag cttcccgatg
      661 agtttgtagt catcgaacgt aagaagcgta gtctctcgac taataccagt gatatttctg
      721 tgacggccac taacgacagc cgcctgtatc cgggagccct gttggtagtg gatgagacct
      781 tgctggagaa taatcctacg ttacttgccg tagatcgtgc cccgatgacg tattcaattg
      841 atctgccagg tttggcttca tccgattcgt tcttacaagt ggaagacccg agcaattctt
      901 ctgtacgcgg tgcggtaaac gatctgctgg cgaagtggca tcaagattat ggtcaggtta
      961 ataatgttcc agcccgcatg cagtatgaaa agatcacggc tcacagtatg gaacaactga
     1021 aggtgaagtt tggatcagac tttgaaaaga ccggaaattc gctggatatt gattttaact
     1081 cagtgcattc cggagagaag cagattcaga ttgttaattt caagcagatt tattataccg
     1141 tgagcgttga cgcagtgaag aatccgggtg atgtgtttca agatacagtc actgttgagg
     1201 atttaaaaca gcgtggtatt tccgcagagc gtcctctcgt ttatattagc agtgtggcgt
     1261 atggtcgcca agtatatttg aagttagaaa caacgtccaa gtctgatgaa gtagaggcgg
     1321 catttgaagc gctcatcaaa ggggtcaagg tggccccaca gaccgagtgg gcggcgattt
     1381 tggccgctac ggaagtgaag gcggttattc tgggtggcga cccttcgtcc ggggcccgcg
     1441 tcgtaacagg caaggtggat atggtggaag acttgattca ggaaggatcc cgctttaccg
     1501 cggatcatcc tggtcttccc atttcgtata ctaccagttt tcttcgtgac aatgtggtgg
     1561 ccacctttca aaactctacc gactatgtgg agacgaaggt caccgcctac cgtaacggtg
     1621 atctccttct ggatcattcg ggcgcatatg tagcgcaata ttatattact tgggatgaac
     1681 tgagttatga tcatcaaggc aaggaagtac ttactcccaa ggcatgggac cgtaatggcc
     1741 aggatcttac ggcgcacttt actaccagca ttcctctcaa aggtaatgtg cgtaatctta
     1801 gtgttaaaat tcgtgagtgt accggacttg cctgggaatg gtggcgtacc gtttatgaga
     1861 aaaccgatct cccactggtc cgtaagcgta caattagtat ttggggaact acactgtatc
     1921 ctcaggtaga ggataaggtt gaaaatgact aattcgaagc ttggctgttt tggcggatga
     1981 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
     2041 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
     2101 aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag
     2161 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     2221 gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa
     2281 gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa
     2341 gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt
     2401 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2461 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2521 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2581 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2641 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2701 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     2761 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2821 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2881 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2941 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3001 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3061 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3121 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3181 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3241 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3301 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3361 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3421 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3481 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3541 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3601 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3661 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3721 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3781 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3841 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3901 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3961 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4021 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4081 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4141 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4201 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4261 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4321 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4381 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4441 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4501 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4561 gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat gcatttacgt tgacaccatc
     4621 gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     4681 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     4741 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     4801 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     4861 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     4921 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     4981 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     5041 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     5101 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     5161 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     5221 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     5281 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     5341 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     5401 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     5461 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     5521 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     5581 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     5641 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     5701 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     5761 agcgggcagt gagcgcaacg caattaatgt aagtt
//
