LOCUS       sequence-322428-        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-no charge
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(52..57,58..74,75..81)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    89..105
                     /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     94..116
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    122..191
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             242..268
                     /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             284..301
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             305..337
                     /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(341..349,350..364)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1925..1942)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2095..2181
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2273..2300
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2319..2410
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2411..2479,2480..3271)
                     /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(2629..2648)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3442..4030
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3931..3950
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4184..4201
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4216..4356
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4282..4301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4442..4464
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4542..4619
                     /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             4620..5702
                     /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(4639..4658)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt
       61 aatcatccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      121 agcgccgctg agaaaaagcg aagcggcact gctctttaac aatttatcag acaatctgtg
      181 tgggcactcg accggaatta tcgattaact ttattattaa aaattaaaga ggtatatatt
      241 aatgtatcga ttaaataagg aggaataaac catggggggt tctcatcatc atcatcatca
      301 tggtatggct agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga
      361 taaggatcga tggggatccg aaaccccgac gaaaccgaaa gcggcccaga ccgaaaagaa
      421 aaccgagaaa aaaccggaaa acagcaattc tgaagcagct aaaaaagcgc tgaacgatta
      481 tatttggggc ctgcagtacg acaaactgaa catcctgacc catcaaggtg aaaaactgaa
      541 aaatcatagc tctcgtgaag cctttcaccg tccgggcgaa tacgtggtta tcgaaaagaa
      601 aaaacagagt atctccaacg cgacctcgaa actgagcgtg agttccgcca atgatgaccg
      661 tatttttccg ggcgcgctgc tgaaagctga tcaaagcctg ctggaaaacc tgccgaccct
      721 gattccggtc aatcgcggta aaaccacgat ctcagtgaac ctgccgggcc tgaaaaacgg
      781 tgaatcgaat ctgacggtcg aaaacccgtc aaattcgacc gtgcgtacgg cggttaacaa
      841 tctggtcgaa aaatggattc agaactacag taaaacccat gcagtgccgg ctcgcatgca
      901 gtacgaaagc atctctgcac aaagtatgtc ccagctgcaa gcgaaatttg gcgccgattt
      961 cagcaaagtg ggtgctccgc tgaacgtgga cttttcatcg gttcacaaag gcgaaaaaca
     1021 ggttttcatc gcaaacttcc gtcaagttta ctacaccgct tctgtcgata gtccgaacag
     1081 cccgtcggcg ctgttcggca gtggtattac cccgacggac ctgatcaacc gtggcgttaa
     1141 ttccaaaacc ccgccggtct atgtgtccaa tgtgagctat ggccgcgcga tgtacgttaa
     1201 atttgaaacc acgagcaaat ctaccaaagt gcaggcggcc atcgatgccg tcgtgaaagg
     1261 cgcaaaactg aaagctggta cggaatacgc agccatcctg gcagccacca aaatcacggc
     1321 agttgtcctg ggcggtaatc cgggcgaagc cagcaaagtt attaccggta atatcgatac
     1381 gctgaaagac ctgattcaga aaggttcgaa ctttagcgcg caatctccgg ccgttccgat
     1441 cagttatacc acgtccttcg tcaaagataa tagcattgca accatccaga acaatacgga
     1501 ctatattgaa accaaagtga cgtcttacaa agatggcgct ctgaccctga accatgacgg
     1561 tgcgtttgtc gcccgcttct atgtgtactg ggaagaactg ggccacgatg cagacggtta
     1621 tgaaaccatt cgtagtcgct cctggtcagg caacggttat aatcgtggcg cccattacag
     1681 caccacgctg cgctttaagg gtaacgttcg taatatccgc gttaaagtcc tgggcgcaac
     1741 cggtctggct tgggaaccgt ggcgtctgat ttacagcaaa aacgatctgc cgctggtgcc
     1801 gcagcgcaat atctctacct ggggcaccac gctgcacccg caattcgaag ataaagtggt
     1861 taaagataac acggactaag aattcgaagc ttggctgttt tggcggatga gagaagattt
     1921 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
     1981 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     2041 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     2101 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     2161 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     2221 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc
     2281 atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca
     2341 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2401 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2461 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2521 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2581 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2641 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2701 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2761 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2821 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2881 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2941 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3001 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3061 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3121 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3181 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3241 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3301 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3361 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3421 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3481 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3541 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3601 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3661 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3721 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3781 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3841 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3901 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3961 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4021 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4081 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4141 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4201 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4261 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4321 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4381 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4441 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag
     4501 atcaattcgc gcgcgaaggc gaagcggcat gcatttacgt tgacaccatc gaatggtgca
     4561 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg
     4621 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
     4681 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
     4741 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
     4801 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
     4861 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
     4921 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
     4981 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     5041 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     5101 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     5161 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     5221 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     5281 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     5341 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     5401 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
     5461 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     5521 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     5581 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     5641 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     5701 gagcgcaacg caattaatgt aagttagcgc gaattgatct ggtttgacag cttatcatcg
     5761 actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg
     5821 tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa
     5881 tg
//
