LOCUS       sequence-322528-        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-N342W
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(100..105,106..122,123..129)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    137..153
                     /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     142..164
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    170..239
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             290..316
                     /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             332..349
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             353..385
                     /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(389..397,398..412)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1973..1990)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2143..2229
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2321..2348
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2367..2458
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2459..2527,2528..3319)
                     /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(2677..2696)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3490..4078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3979..3998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4232..4249
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4264..4404
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4330..4349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4490..4512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4590..4667
                     /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             4668..5750
                     /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(4687..4706)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg
       61 acatcataac ggttctggca aatattctga aatgagctgt tgacaattaa tcatccggct
      121 cgtataatgt gtggaattgt gagcggataa caatttcaca caggaaacag cgccgctgag
      181 aaaaagcgaa gcggcactgc tctttaacaa tttatcagac aatctgtgtg ggcactcgac
      241 cggaattatc gattaacttt attattaaaa attaaagagg tatatattaa tgtatcgatt
      301 aaataaggag gaataaacca tggggggttc tcatcatcat catcatcatg gtatggctag
      361 catgactggt ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcgatg
      421 gggatccgaa accccgacga aaccgaaagc ggcccagacc gaaaagaaaa ccgagaaaaa
      481 accggaaaac agcaattctg aagcagctaa aaaagcgctg aacgattata tttggggcct
      541 gcagtacgac aaactgaaca tcctgaccca tcaaggtgaa aaactgaaaa atcatagctc
      601 tcgtgaagcc tttcaccgtc cgggcgaata cgtggttatc gaaaagaaaa aacagagtat
      661 ctccaacgcg acctcgaaac tgagcgtgag ttccgccaat gatgaccgta tttttccggg
      721 cgcgctgctg aaagctgatc aaagcctgct ggaaaacctg ccgaccctga ttccggtcaa
      781 tcgcggtaaa accacgatct cagtgaacct gccgggcctg aaaaacggtg aatcgaatct
      841 gacggtcgaa aacccgtcaa attcgaccgt gcgtacggcg gttaacaatc tggtcgaaaa
      901 atggattcag aactacagta aaacccatgc agtgccggct cgcatgcagt acgaaagcat
      961 ctctgcacaa agtatgtccc agctgcaatg caaatttggc gccgatttca gcaaagtggg
     1021 tgctccgctg aacgtggact tttcatcggt tcacaaaggc gaaaaacagg ttttcatcgc
     1081 aaacttccgt caagtttact acaccgcttc tgtcgatagt ccgaacagcc cgtcggcgct
     1141 gttcggcagt ggtattaccc cgacggacct gatcaaccgt ggcgttaatt ccaaaacccc
     1201 gccggtctat gtgtccaatg tgagctatgg ccgcgcgatg tacgttaaat ttgaaaccac
     1261 gagcaaatct accaaagtgc aggcggccat cgatgccgtc gtgaaaggcg caaaactgaa
     1321 agctggtacg gaatacgaaa acatcctgaa atggaccaaa atcacggcag ttgtcctggg
     1381 cggtaatccg ggcgaagcca gcaaagttat taccggtaat atcgatacgc tgaaagacct
     1441 gattcagaaa ggttcgaact ttagcgcgca atctccggcc gttccgatca gttataccac
     1501 gtccttcgtc aaagataata gcattgcaac catccagtgc aatacggact atattgaaac
     1561 caaagtgacg tcttacaaag atggcgctct gaccctgaac catgacggtg cgtttgtcgc
     1621 ccgcttctat gtgtactggg aagaactggg ccacgatgca gacggttatg aaaccattcg
     1681 tagtcgctcc tggtcaggca acggttataa tcgtggcgcc cattacagca ccacgctgcg
     1741 ctttaagggt aacgttcgta atatccgcgt taaagtcctg ggcgcaaccg gtctggcttg
     1801 ggaaccgtgg cgtctgattt acagcaaaaa cgatctgccg ctggtgccgc agcgcaatat
     1861 ctctacctgg ggcaccacgc tgcacccgca attcgaagat aaagtggtta aagataacac
     1921 ggactaagaa ttcgaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac
     1981 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg
     2041 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
     2101 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     2161 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     2221 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     2281 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
     2341 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
     2401 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2461 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2521 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2581 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2641 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2701 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2761 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2821 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2881 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2941 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3001 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3061 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3121 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3181 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3241 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3301 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3361 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3421 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3481 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3541 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3601 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3661 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3721 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3781 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3841 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3901 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3961 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4021 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4081 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4141 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4201 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4261 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     4321 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4381 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4441 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4501 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat caattcgcgc
     4561 gcgaaggcga agcggcatgc atttacgttg acaccatcga atggtgcaaa acctttcgcg
     4621 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     4681 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     4741 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     4801 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     4861 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     4921 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     4981 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     5041 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     5101 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     5161 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     5221 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     5281 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     5341 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     5401 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     5461 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     5521 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     5581 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     5641 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     5701 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     5761 attaatgtaa gttagcgcga attgatctgg tttgacagct tatcatcgac tgcacggtgc
     5821 accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa
     5881 at
//
