LOCUS       sequence-448044-        4461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses T4 lysozyme in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4461)
  AUTHORS   Toro TB, Nguyen TP, Watt TJ
  TITLE     An improved 96-well turbidity assay for T4 lysozyme activity.
  JOURNAL   MethodsX. 2015 May 18;2:256-62. doi: 10.1016/j.mex.2015.05.004. 
            eCollection 2015.
  PUBMED    26150996
REFERENCE   2  (bases 1 to 4461)
  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: "MethodsX.";
            date: "2015-05-18"; volume: "2:256-62. doi"; pages: " 
            10.1016/j.mex.2015.05.004. eCollection 2015"
COMMENT     Sequence Label: pJExpress401-T4 Lysozyme C54T C97A (WT*)
FEATURES             Location/Qualifiers
     source          1..4461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1417..1433
                     /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)."
     promoter        join(1458..1472,1473..1478,1479..1495,1496..1502)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1478..1494
                     /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)."
     protein_bind    1508..1527
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     CDS             2066..2086
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2087..2104
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2309..2328)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2395..2486
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2487..3296
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3379..4461
                     /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(3398..3417)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatgcatt tagaataaat tttgtgtcgc ccttaattgt gagcggataa caattacgag
     1441 cttcatgcac agtgaaatca tgaaaaattt atttgctttg tgagcggata acaattataa
     1501 tatgtggaat tgtgagcgct cacaattcca caacggtttc cctctagaaa taattttgtt
     1561 taacttttag gaggtaaaac atatgaacat ttttgagatg ctgagaattg atgagggctt
     1621 acgtttgaga atctatcgcg acaccgaagg ttactacacc atcggcattg gccatttgct
     1681 gacgcgctct ccaagcttaa atgcggccaa atcggagctg gatcgcgcaa ttggccgtaa
     1741 tacgaatggt gtcatcactc gtgacgaagc tgagcgtctg tttaatcagg acgttgacgc
     1801 agcggtccgc ggtattctgc gtaatgcgcg cctgcgccca gtgtacgata gcctggacgc
     1861 cgtgcgtcgt gcggcactga ttaacatggt tttccaaatg ggtgaaacgg gtgtggcggg
     1921 tttcaccaac tccctgcgta tgctgcaaca acgtcgttgg gatgaggcgg cagttaacct
     1981 ggctcgtagc cgttggtaca accagacccc gaaccgtgcg cgtcgcgtca ttacgacctt
     2041 tcgtaccggc acctgggatg cctatgaaaa tctgtatttc cagggtcacc atcaccacca
     2101 tcactaactc gagccccaag ggcgacaccc cctaattagc ccgggcgaaa ggcccagtct
     2161 ttcgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg catggggagt
     2221 ccccacacta ccatcggcgc tacggcgttt cacttctgag ttcggcatgg ggtcaggtgg
     2281 gaccaccgcg ctactgccgc caggcaaaca aggggtgtta tgagccatat tcaggtataa
     2341 atgggctcgc gataatgttc agaattggtt aattggttgt aacactgacc cctatttgtt
     2401 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2461 ttcaataata ttgaaaaagg aagaatatga gccatattca acgggaaacg tcgaggccgc
     2521 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     2581 ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc
     2641 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     2701 ggctgacgga atttatgcca cttccgacca tcaagcattt tatccgtact cctgatgatg
     2761 catggttact caccactgcg atccccggaa aaacagcgtt ccaggtatta gaagaatatc
     2821 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcactcga
     2881 ttcctgtttg taattgtcct tttaacagtg atcgcgtatt tcgcctcgct caggcgcaat
     2941 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     3001 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     3061 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     3121 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     3181 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     3241 ataatcctga tatgaataaa ttgcaatttc atttgatgct cgatgagttt ttctaagcgg
     3301 cgcgccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     3361 caattcaggg tggtgaatat gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     3421 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     3481 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     3541 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     3601 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     3661 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     3721 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     3781 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     3841 cccatcaaca gtattatttt ctcccatgag gacggtacgc gactgggcgt ggagcatctg
     3901 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     3961 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     4021 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     4081 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     4141 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     4201 gataccgaag atagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     4261 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     4321 ggcaatcagc tgttgccagt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     4381 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4441 cgactggaaa gcgggcagtg a
//
