LOCUS       sequence-322432-        5882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of inserted ILY variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5882)
  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 5882)
  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-ILY-WT-N338W
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(302..307,308..324,325..331)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    339..355
                     /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     344..366
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    372..441
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             492..518
                     /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             534..551
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             555..587
                     /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(591..599,600..614)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(2175..2192)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2345..2431
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2523..2550
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2569..2660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2661..2729,2730..3521)
                     /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(2879..2898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3692..4280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4181..4200
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4434..4451
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4466..4606
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4532..4551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4692..4714
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4792..4869
                     /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             join(4870..5882,1..70)
                     /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(4889..4908)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
       61 cgggcagtga gcgcaacgca attaatgtaa gttagcgcga attgatctgg tttgacagct
      121 tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
      181 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
      241 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
      301 gttgacaatt aatcatccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      361 cacaggaaac agcgccgctg agaaaaagcg aagcggcact gctctttaac aatttatcag
      421 acaatctgtg tgggcactcg accggaatta tcgattaact ttattattaa aaattaaaga
      481 ggtatatatt aatgtatcga ttaaataagg aggaataaac catggggggt tctcatcatc
      541 atcatcatca tggtatggct agcatgactg gtggacagca aatgggtcgg gatctgtacg
      601 acgatgacga taaggatcga tggggatccg aaaccccgac gaaaccgaaa gcggcccaga
      661 ccgaaaagaa aaccgagaaa aaaccggaaa acagcaattc tgaagcagct aaaaaagcgc
      721 tgaacgatta tatttggggc ctgcagtacg acaaactgaa catcctgacc catcaaggtg
      781 aaaaactgaa aaatcatagc tctcgtgaag cctttcaccg tccgggcgaa tacgtggtta
      841 tcgaaaagaa aaaacagagt atctccaacg cgacctcgaa actgagcgtg agttccgcca
      901 atgatgaccg tatttttccg ggcgcgctgc tgaaagctga tcaaagcctg ctggaaaacc
      961 tgccgaccct gattccggtc aatcgcggta aaaccacgat ctcagtgaac ctgccgggcc
     1021 tgaaaaacgg tgaatcgaat ctgacggtcg aaaacccgtc aaattcgacc gtgcgtacgg
     1081 cggttaacaa tctggtcgaa aaatggattc agaactacag taaaacccat gcagtgccgg
     1141 ctcgcatgca gtacgaaagc atctctgcac aaagtatgtc ccagctgcaa gcgaaatttg
     1201 gcgccgattt cagcaaagtg ggtgctccgc tgaacgtgga cttttcatcg gttcacaaag
     1261 gcgaaaaaca ggttttcatc gcaaacttcc gtcaagttta ctacaccgct tctgtcgata
     1321 gtccgaacag cccgtcggcg ctgttcggca gtggtattac cccgacggac ctgatcaacc
     1381 gtggcgttaa ttccaaaacc ccgccggtct atgtgtccaa tgtgagctat ggccgcgcga
     1441 tgtacgttaa atttgaaacc acgagcaaat ctaccaaagt gcaggcggcc atcgatgccg
     1501 tcgtgaaagg cgcaaaactg aaagctggta cggaatacga atggatcctg aaaaacacca
     1561 aaatcacggc agttgtcctg ggcggtaatc cgggcgaagc cagcaaagtt attaccggta
     1621 atatcgatac gctgaaagac ctgattcaga aaggttcgaa ctttagcgcg caatctccgg
     1681 ccgttccgat cagttatacc acgtccttcg tcaaagataa tagcattgca accatccaga
     1741 acaatacgga ctatattgaa accaaagtga cgtcttacaa agatggcgct ctgaccctga
     1801 accatgacgg tgcgtttgtc gcccgcttct atgtgtactg ggaagaactg ggccacgatg
     1861 cagacggtta tgaaaccatt cgtagtcgct cctggtcagg caacggttat aatcgtggcg
     1921 cccattacag caccacgctg cgctttaagg gtaacgttcg taatatccgc gttaaagtcc
     1981 tgggcgcaac cggtctggct tgggaaccgt ggcgtctgat ttacagcaaa aacgatctgc
     2041 cgctggtgcc gcagcgcaat atctctacct ggggcaccac gctgcacccg caattcgaag
     2101 ataaagtggt taaagataac acggactaag aattcgaagc ttggctgttt tggcggatga
     2161 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
     2221 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
     2281 aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag
     2341 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     2401 gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa
     2461 gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa
     2521 gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt
     2581 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2641 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2701 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2761 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2821 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2881 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     2941 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3001 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3061 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3121 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3181 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3241 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3301 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3361 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3421 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3481 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3541 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3601 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3661 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3721 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3781 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3841 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3901 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3961 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4021 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4081 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4141 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4201 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4261 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4321 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4381 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4441 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4501 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4561 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4621 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4681 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4741 gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat gcatttacgt tgacaccatc
     4801 gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     4861 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     4921 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     4981 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     5041 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     5101 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     5161 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     5221 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     5281 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     5341 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     5401 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     5461 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     5521 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     5581 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     5641 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     5701 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     5761 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     5821 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     5881 tc
//
