LOCUS       sequence-322554-        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-HB2lock-Y336W
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(86..91,92..108,109..115)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    123..139
                     /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     128..150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    156..225
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             276..302
                     /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             318..335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             339..371
                     /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(375..383,384..398)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1959..1976)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2129..2215
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2307..2334
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2353..2444
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2445..2513,2514..3305)
                     /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(2663..2682)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3476..4064
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3965..3984
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4218..4235
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4250..4390
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4316..4335)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4476..4498
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4576..4653
                     /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             4654..5736
                     /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(4673..4692)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
       61 ctggcaaata ttctgaaatg agctgttgac aattaatcat ccggctcgta taatgtgtgg
      121 aattgtgagc ggataacaat ttcacacagg aaacagcgcc gctgagaaaa agcgaagcgg
      181 cactgctctt taacaattta tcagacaatc tgtgtgggca ctcgaccgga attatcgatt
      241 aactttatta ttaaaaatta aagaggtata tattaatgta tcgattaaat aaggaggaat
      301 aaaccatggg gggttctcat catcatcatc atcatggtat ggctagcatg actggtggac
      361 agcaaatggg tcgggatctg tacgacgatg acgataagga tcgatgggga tccgaaaccc
      421 cgacgaaacc gaaagcggcc cagaccgaaa agaaaaccga gaaaaaaccg gaaaacagca
      481 attctgaagc agctaaaaaa gcgctgaacg attatatttg gggcctgcag tacgacaaac
      541 tgaacatcct gacccatcaa ggtgaaaaac tgaaaaatca tagctctcgt gaagcctttc
      601 accgtccggg cgaatacgtg gttatcgaaa agaaaaaaca gagtatctcc aacgcgacct
      661 cgaaactgag cgtgagttcc gccaatgatg accgtatttt tccgggcgcg ctgctgaaag
      721 ctgatcaaag cctgctggaa aacctgccga ccctgattcc ggtcaatcgc ggtaaaacca
      781 cgatctcagt gaacctgccg ggcctgaaaa acggtgaatc gaatctgacg gtcgaaaacc
      841 cgtcaaattc gaccgtgcgt acggcggtta acaatctggt cgaaaaatgg attcagaact
      901 acagtaaaac ccatgcagtg ccggctcgca tgcagtgcga aagcatctct gcacaaagta
      961 tgtcccagct gcaagcgaaa tttggcgccg atttcagcaa agtgggtgct ccgctgaacg
     1021 tggacttttc atcggttcac aaaggcgaaa aacaggtttt catcgcaaac ttccgtcaag
     1081 tttactacac cgcttctgtc gatagtccga acagcccgtc ggcgctgttc ggcagtggta
     1141 ttaccccgac ggacctgatc aaccgtggcg ttaattccaa aaccccgccg gtctatgtgt
     1201 ccaatgtgag ctatggccgc gcgatgtacg ttaaatttga aaccacgagc aaatctacca
     1261 aagtgcaggc ggccatctgt gccgtcgtga aaggcgcaaa actgaaagct ggtacggaat
     1321 gggaaaacat cctgaaaaac accaaaatca cggcagttgt cctgggcggt aatccgggcg
     1381 aagccagcaa agttattacc ggtaatatcg atacgctgaa agacctgatt cagaaaggtt
     1441 cgaactttag cgcgcaatct ccggccgttc cgatcagtta taccacgtcc ttcgtcaaag
     1501 ataatagcat tgcaaccatc cagaacaata cggactatat tgaaaccaaa gtgacgtctt
     1561 acaaagatgg cgctctgacc ctgaaccatg acggtgcgtt tgtcgcccgc ttctatgtgt
     1621 actgggaaga actgggccac gatgcagacg gttatgaaac cattcgtagt cgctcctggt
     1681 caggcaacgg ttataatcgt ggcgcccatt acagcaccac gctgcgcttt aagggtaacg
     1741 ttcgtaatat ccgcgttaaa gtcctgggcg caaccggtct ggcttgggaa ccgtggcgtc
     1801 tgatttacag caaaaacgat ctgccgctgg tgccgcagcg caatatctct acctggggca
     1861 ccacgctgca cccgcaattc gaagataaag tggttaaaga taacacggac taagaattcg
     1921 aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
     1981 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct
     2041 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc
     2101 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     2161 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     2221 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     2281 ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt
     2341 tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2401 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2461 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2521 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2581 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2641 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     2701 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2761 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2821 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2881 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2941 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3001 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3061 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3121 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3181 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3241 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3301 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3361 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3421 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3481 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3541 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3601 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3661 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3721 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3781 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3841 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3901 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3961 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4021 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4081 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4141 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4201 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4261 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     4321 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4381 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4441 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4501 ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg
     4561 gcatgcattt acgttgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag
     4621 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     4681 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     4741 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     4801 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     4861 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     4921 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     4981 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     5041 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     5101 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     5161 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     5221 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     5281 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     5341 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     5401 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     5461 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     5521 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     5581 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     5641 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5701 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     5761 gcgcgaattg atctggtttg acagcttatc atcgactgca cggtgcacca atgcttctgg
     5821 cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt
     5881 cg
//
