LOCUS       sequence-322469-        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-HB1lock-no charge
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(92..97,98..114,115..121)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    129..145
                     /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     134..156
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    162..231
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             282..308
                     /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             324..341
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             345..377
                     /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(381..389,390..404)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1965..1982)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2135..2221
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2313..2340
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2359..2450
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2451..2519,2520..3311)
                     /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(2669..2688)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3482..4070
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3971..3990
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4224..4241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4256..4396
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4322..4341)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4482..4504
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4582..4659
                     /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             4660..5742
                     /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(4679..4698)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata
       61 acggttctgg caaatattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat
      121 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agcgccgctg agaaaaagcg
      181 aagcggcact gctctttaac aatttatcag acaatctgtg tgggcactcg accggaatta
      241 tcgattaact ttattattaa aaattaaaga ggtatatatt aatgtatcga ttaaataagg
      301 aggaataaac catggggggt tctcatcatc atcatcatca tggtatggct agcatgactg
      361 gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatcga tggggatccg
      421 aaaccccgac gaaaccgaaa gcggcccaga ccgaaaagaa aaccgagaaa aaaccggaaa
      481 acagcaattc tgaagcagct aaaaaagcgc tgaacgatta tatttggggc ctgcagtacg
      541 acaaactgaa catcctgacc catcaaggtg aaaaactgaa aaatcatagc tctcgtgaag
      601 cctttcaccg tccgggcgaa tacgtggtta tcgaaaagaa aaaacagagt atctccaacg
      661 cgacctcgaa actgagcgtg agttccgcca atgatgaccg tatttttccg ggcgcgctgc
      721 tgaaagctga tcaaagcctg ctggaaaacc tgccgaccct gattccggtc aatcgcggta
      781 aaaccacgat ctcagtgaac ctgccgggcc tgaaaaacgg tgaatcgaat ctgacggtcg
      841 aaaacccgtc aaattcgacc gtgcgtacgg cggttaacaa tctggtcgaa aaatggattc
      901 agaactacag taaaacccat gcagtgccgg ctcgcatgca gtacgaaagc atctctgcac
      961 aaagtatgtc ccagctgcaa tgcaaatttg gcgccgattt cagcaaagtg ggtgctccgc
     1021 tgaacgtgga cttttcatcg gttcacaaag gcgaaaaaca ggttttcatc gcaaacttcc
     1081 gtcaagttta ctacaccgct tctgtcgata gtccgaacag cccgtcggcg ctgttcggca
     1141 gtggtattac cccgacggac ctgatcaacc gtggcgttaa ttccaaaacc ccgccggtct
     1201 atgtgtccaa tgtgagctat ggccgcgcga tgtacgttaa atttgaaacc acgagcaaat
     1261 ctaccaaagt gcaggcggcc atcgatgccg tcgtgaaagg cgcaaaactg aaagctggta
     1321 cggaatacgc agccatcctg gcagccacca aaatcacggc agttgtcctg ggcggtaatc
     1381 cgggcgaagc cagcaaagtt attaccggta atatcgatac gctgaaagac ctgattcaga
     1441 aaggttcgaa ctttagcgcg caatctccgg ccgttccgat cagttatacc acgtccttcg
     1501 tcaaagataa tagcattgca accatccagt gcaatacgga ctatattgaa accaaagtga
     1561 cgtcttacaa agatggcgct ctgaccctga accatgacgg tgcgtttgtc gcccgcttct
     1621 atgtgtactg ggaagaactg ggccacgatg cagacggtta tgaaaccatt cgtagtcgct
     1681 cctggtcagg caacggttat aatcgtggcg cccattacag caccacgctg cgctttaagg
     1741 gtaacgttcg taatatccgc gttaaagtcc tgggcgcaac cggtctggct tgggaaccgt
     1801 ggcgtctgat ttacagcaaa aacgatctgc cgctggtgcc gcagcgcaat atctctacct
     1861 ggggcaccac gctgcacccg caattcgaag ataaagtggt taaagataac acggactaag
     1921 aattcgaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     1981 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     2041 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     2101 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     2161 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     2221 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     2281 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     2341 tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2401 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2461 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2521 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2581 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2641 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg
     2701 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2761 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2821 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2881 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2941 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3001 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3061 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3121 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3181 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3241 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3301 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3361 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3421 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3481 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3541 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3601 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3661 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3721 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3781 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3841 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3901 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3961 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4021 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4081 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4141 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4201 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4261 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     4321 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4381 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4441 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4501 gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc
     4561 gaagcggcat gcatttacgt tgacaccatc gaatggtgca aaacctttcg cggtatggca
     4621 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
     4681 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
     4741 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     4801 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     4861 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     4921 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     4981 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     5041 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     5101 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
     5161 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     5221 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     5281 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     5341 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     5401 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     5461 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     5521 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     5581 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     5641 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5701 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     5761 aagttagcgc gaattgatct ggtttgacag cttatcatcg actgcacggt gcaccaatgc
     5821 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
     5881 at
//
