LOCUS       sequence-322465-        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-charge+
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(157..162,163..179,180..186)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    194..210
                     /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     199..221
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    227..296
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             347..373
                     /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             389..406
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             410..442
                     /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(446..454,455..469)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(2030..2047)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2200..2286
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2378..2405
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2424..2515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2516..2584,2585..3376)
                     /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(2734..2753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3547..4135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4036..4055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4289..4306
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4321..4461
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4387..4406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4547..4569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4647..4724
                     /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             4725..5807
                     /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(4744..4763)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac
       61 tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca
      121 tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca tccggctcgt
      181 ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcgc cgctgagaaa
      241 aagcgaagcg gcactgctct ttaacaattt atcagacaat ctgtgtgggc actcgaccgg
      301 aattatcgat taactttatt attaaaaatt aaagaggtat atattaatgt atcgattaaa
      361 taaggaggaa taaaccatgg ggggttctca tcatcatcat catcatggta tggctagcat
      421 gactggtgga cagcaaatgg gtcgggatct gtacgacgat gacgataagg atcgatgggg
      481 atccgaaacc ccgacgaaac cgaaagcggc ccagaccgaa aagaaaaccg agaaaaaacc
      541 ggaaaacagc aattctgaag cagctaaaaa agcgctgaac gattatattt ggggcctgca
      601 gtacgacaaa ctgaacatcc tgacccatca aggtgaaaaa ctgaaaaatc atagctctcg
      661 tgaagccttt caccgtccgg gcgaatacgt ggttatcgaa aagaaaaaac agagtatctc
      721 caacgcgacc tcgaaactga gcgtgagttc cgccaatgat gaccgtattt ttccgggcgc
      781 gctgctgaaa gctgatcaaa gcctgctgga aaacctgccg accctgattc cggtcaatcg
      841 cggtaaaacc acgatctcag tgaacctgcc gggcctgaaa aacggtgaat cgaatctgac
      901 ggtcgaaaac ccgtcaaatt cgaccgtgcg tacggcggtt aacaatctgg tcgaaaaatg
      961 gattcagaac tacagtaaaa cccatgcagt gccggctcgc atgcagtacg aaagcatctc
     1021 tgcacaaagt atgtcccagc tgcaatgcaa atttggcgcc gatttcagca aagtgggtgc
     1081 tccgctgaac gtggactttt catcggttca caaaggcgaa aaacaggttt tcatcgcaaa
     1141 cttccgtcaa gtttactaca ccgcttctgt cgatagtccg aacagcccgt cggcgctgtt
     1201 cggcagtggt attaccccga cggacctgat caaccgtggc gttaattcca aaaccccgcc
     1261 ggtctatgtg tccaatgtga gctatggccg cgcgatgtac gttaaatttg aaaccacgag
     1321 caaatctacc aaagtgcagg cggccatcga tgccgtcgtg aaaggcgcaa aactgaaagc
     1381 tggtacggaa aaggaaaaca tcaagaaaaa caccaaaatc acggcagttg tcctgggcgg
     1441 taatccgggc gaagccagca aagttattac cggtaatatc gatacgctga aagacctgat
     1501 tcagaaaggt tcgaacttta gcgcgcaatc tccggccgtt ccgatcagtt ataccacgtc
     1561 cttcgtcaaa gataatagca ttgcaaccat ccagtgcaat acggactata ttgaaaccaa
     1621 agtgacgtct tacaaagatg gcgctctgac cctgaaccat gacggtgcgt ttgtcgcccg
     1681 cttctatgtg tactgggaag aactgggcca cgatgcagac ggttatgaaa ccattcgtag
     1741 tcgctcctgg tcaggcaacg gttataatcg tggcgcccat tacagcacca cgctgcgctt
     1801 taagggtaac gttcgtaata tccgcgttaa agtcctgggc gcaaccggtc tggcttggga
     1861 accgtggcgt ctgatttaca gcaaaaacga tctgccgctg gtgccgcagc gcaatatctc
     1921 tacctggggc accacgctgc acccgcaatt cgaagataaa gtggttaaag ataacacgga
     1981 ctaagaattc gaagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga
     2041 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     2101 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     2161 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     2221 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     2281 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     2341 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     2401 ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc
     2461 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2521 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2581 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2641 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2701 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     2761 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2821 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2881 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2941 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3001 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3061 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3121 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3181 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3241 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3301 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3361 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3421 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3481 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3541 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3601 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3661 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3721 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3781 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3841 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3901 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3961 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4021 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4081 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4141 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4201 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4261 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4321 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     4381 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4441 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4501 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4561 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg
     4621 aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc tttcgcggta
     4681 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4741 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4801 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4861 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4921 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4981 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5041 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5101 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5161 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     5221 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5281 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5341 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5401 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5461 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5521 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     5581 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     5641 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     5701 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5761 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5821 aatgtaagtt agcgcgaatt gatctggttt gacagcttat catcgactgc acggtgcacc
     5881 aa
//
