LOCUS       sequence-322552-        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-no charge
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(75..80,81..97,98..104)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    112..128
                     /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     117..139
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    145..214
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             265..291
                     /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             307..324
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             328..360
                     /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(364..372,373..387)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1948..1965)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2118..2204
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2296..2323
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2342..2433
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2434..2502,2503..3294)
                     /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(2652..2671)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3465..4053
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3954..3973
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4207..4224
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4239..4379
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4305..4324)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4465..4487
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4565..4642
                     /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             4643..5725
                     /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(4662..4681)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat
       61 tctgaaatga gctgttgaca attaatcatc cggctcgtat aatgtgtgga attgtgagcg
      121 gataacaatt tcacacagga aacagcgccg ctgagaaaaa gcgaagcggc actgctcttt
      181 aacaatttat cagacaatct gtgtgggcac tcgaccggaa ttatcgatta actttattat
      241 taaaaattaa agaggtatat attaatgtat cgattaaata aggaggaata aaccatgggg
      301 ggttctcatc atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt
      361 cgggatctgt acgacgatga cgataaggat cgatggggat ccgaaacccc gacgaaaccg
      421 aaagcggccc agaccgaaaa gaaaaccgag aaaaaaccgg aaaacagcaa ttctgaagca
      481 gctaaaaaag cgctgaacga ttatatttgg ggcctgcagt acgacaaact gaacatcctg
      541 acccatcaag gtgaaaaact gaaaaatcat agctctcgtg aagcctttca ccgtccgggc
      601 gaatacgtgg ttatcgaaaa gaaaaaacag agtatctcca acgcgacctc gaaactgagc
      661 gtgagttccg ccaatgatga ccgtattttt ccgggcgcgc tgctgaaagc tgatcaaagc
      721 ctgctggaaa acctgccgac cctgattccg gtcaatcgcg gtaaaaccac gatctcagtg
      781 aacctgccgg gcctgaaaaa cggtgaatcg aatctgacgg tcgaaaaccc gtcaaattcg
      841 accgtgcgta cggcggttaa caatctggtc gaaaaatgga ttcagaacta cagtaaaacc
      901 catgcagtgc cggctcgcat gcagtgcgaa agcatctctg cacaaagtat gtcccagctg
      961 caagcgaaat ttggcgccga tttcagcaaa gtgggtgctc cgctgaacgt ggacttttca
     1021 tcggttcaca aaggcgaaaa acaggttttc atcgcaaact tccgtcaagt ttactacacc
     1081 gcttctgtcg atagtccgaa cagcccgtcg gcgctgttcg gcagtggtat taccccgacg
     1141 gacctgatca accgtggcgt taattccaaa accccgccgg tctatgtgtc caatgtgagc
     1201 tatggccgcg cgatgtacgt taaatttgaa accacgagca aatctaccaa agtgcaggcg
     1261 gccatctgtg ccgtcgtgaa aggcgcaaaa ctgaaagctg gtacggaata cgcagccatc
     1321 ctggcagcca ccaaaatcac ggcagttgtc ctgggcggta atccgggcga agccagcaaa
     1381 gttattaccg gtaatatcga tacgctgaaa gacctgattc agaaaggttc gaactttagc
     1441 gcgcaatctc cggccgttcc gatcagttat accacgtcct tcgtcaaaga taatagcatt
     1501 gcaaccatcc agaacaatac ggactatatt gaaaccaaag tgacgtctta caaagatggc
     1561 gctctgaccc tgaaccatga cggtgcgttt gtcgcccgct tctatgtgta ctgggaagaa
     1621 ctgggccacg atgcagacgg ttatgaaacc attcgtagtc gctcctggtc aggcaacggt
     1681 tataatcgtg gcgcccatta cagcaccacg ctgcgcttta agggtaacgt tcgtaatatc
     1741 cgcgttaaag tcctgggcgc aaccggtctg gcttgggaac cgtggcgtct gatttacagc
     1801 aaaaacgatc tgccgctggt gccgcagcgc aatatctcta cctggggcac cacgctgcac
     1861 ccgcaattcg aagataaagt ggttaaagat aacacggact aagaattcga agcttggctg
     1921 ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     1981 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     2041 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     2101 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     2161 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     2221 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     2281 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     2341 ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2401 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2461 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2521 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2581 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2641 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     2701 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2761 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2821 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2881 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2941 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3001 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3061 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3121 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3181 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3241 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3301 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3361 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3421 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3481 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3541 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3601 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3661 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3721 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3781 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3841 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3901 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3961 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4021 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4081 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4141 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4201 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4261 acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat
     4321 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4381 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4441 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4501 caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg catgcattta
     4561 cgttgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     4621 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     4681 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     4741 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     4801 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     4861 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     4921 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     4981 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     5041 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     5101 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     5161 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     5221 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     5281 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     5341 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     5401 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     5461 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     5521 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     5581 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     5641 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5701 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag cgcgaattga
     5761 tctggtttga cagcttatca tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc
     5821 catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca
     5881 ag
//
