LOCUS       sequence-322453-        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-prepore
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(314..319,320..336,337..343)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    351..367
                     /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     356..378
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    384..453
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             504..530
                     /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             546..563
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             567..599
                     /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(603..611,612..626)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(2187..2204)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2357..2443
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2535..2562
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2581..2672
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2673..2741,2742..3533)
                     /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(2891..2910)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3704..4292
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4193..4212
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4446..4463
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4478..4618
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4544..4563)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4704..4726
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4804..4881
                     /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(4882..5882,1..82)
                     /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(4901..4920)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
       61 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagcgc gaattgatct
      121 ggtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat
      181 cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg
      241 cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt
      301 ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa ttgtgagcgg
      361 ataacaattt cacacaggaa acagcgccgc tgagaaaaag cgaagcggca ctgctcttta
      421 acaatttatc agacaatctg tgtgggcact cgaccggaat tatcgattaa ctttattatt
      481 aaaaattaaa gaggtatata ttaatgtatc gattaaataa ggaggaataa accatggggg
      541 gttctcatca tcatcatcat catggtatgg ctagcatgac tggtggacag caaatgggtc
      601 gggatctgta cgacgatgac gataaggatc gatggggatc cgaaaccccg acgaaaccga
      661 aagcggccca gaccgaaaag aaaaccgaga aaaaaccgga aaacagcaat tctgaagcag
      721 ctaaaaaagc gctgaacgat tatatttggg gcctgcagta cgacaaactg aacatcctga
      781 cccatcaagg tgaaaaactg aaaaatcata gctctcgtga agcctttcac cgtccgggcg
      841 aatacgtggt ttgcgaaaag aaaaaacaga gtatctccaa cgcgacctcg aaactgagcg
      901 tgagttccgc caatgatgac cgtatttttc cgggcgcgct gctgaaagct gatcaaagcc
      961 tgctggaaaa cctgccgacc ctgattccgg tcaatcgcgg taaaaccacg atctcagtga
     1021 acctgccggg cctgaaaaac ggtgaatcga atctgacggt cgaaaacccg tcaaattcga
     1081 ccgtgcgtac ggcggttaac aatctggtcg aaaaatggat tcagaactac agtaaaaccc
     1141 atgcagtgcc ggctcgcatg cagtacgaaa gcatctctgc acaaagtatg tcccagctgc
     1201 aagcgaaatt tggcgccgat ttcagcaaag tgggtgctcc gctgaacgtg gacttttcat
     1261 cggttcacaa atgcgaaaaa caggttttca tcgcaaactt ccgtcaagtt tactacaccg
     1321 cttctgtcga tagtccgaac agcccgtcgg cgctgttcgg cagtggtatt accccgacgg
     1381 acctgatcaa ccgtggcgtt aattccaaaa ccccgccggt ctatgtgtcc aatgtgagct
     1441 atggccgcgc gatgtacgtt aaatttgaaa ccacgagcaa atctaccaaa gtgcaggcgg
     1501 ccatcgatgc cgtcgtgaaa ggcgcaaaac tgaaagctgg tacggaatac gaaaacatcc
     1561 tgaaaaacac caaaatcacg gcagttgtcc tgggcggtaa tccgggcgaa gccagcaaag
     1621 ttattaccgg taatatcgat acgctgaaag acctgattca gaaaggttcg aactttagcg
     1681 cgcaatctcc ggccgttccg atcagttata ccacgtcctt cgtcaaagat aatagcattg
     1741 caaccatcca gaacaatacg gactatattg aaaccaaagt gacgtcttac aaagatggcg
     1801 ctctgaccct gaaccatgac ggtgcgtttg tcgcccgctt ctatgtgtac tgggaagaac
     1861 tgggccacga tgcagacggt tatgaaacca ttcgtagtcg ctcctggtca ggcaacggtt
     1921 ataatcgtgg cgcccattac agcaccacgc tgcgctttaa gggtaacgtt cgtaatatcc
     1981 gcgttaaagt cctgggcgca accggtctgg cttgggaacc gtggcgtctg atttacagca
     2041 aaaacgatct gccgctggtg ccgcagcgca atatctctac ctggggcacc acgctgcacc
     2101 cgcaattcga agataaagtg gttaaagata acacggacta agaattcgaa gcttggctgt
     2161 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     2221 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     2281 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     2341 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     2401 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     2461 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     2521 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
     2581 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2641 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2701 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2761 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2821 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2881 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2941 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3001 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3061 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3121 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3181 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3241 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3301 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3361 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3421 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3481 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3541 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3601 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3661 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3721 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3781 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3841 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3901 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3961 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4021 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4081 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4141 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4201 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4261 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4321 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4381 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4441 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4501 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     4561 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4621 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4681 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4741 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcatttac
     4801 gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     4861 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     4921 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     4981 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     5041 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     5101 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     5161 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     5221 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     5281 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     5341 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     5401 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     5461 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     5521 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     5581 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     5641 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     5701 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     5761 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     5821 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     5881 ac
//
