LOCUS       sequence-371410-        5540 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express phage alpha3 lysis protein E in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5540)
  TITLE     Bacterial expression plasmids for production of phage alpha 3 lysis 
            protein E
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5540)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET29a-alpha-3-E
FEATURES             Location/Qualifiers
     source          1..5540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(209..232)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     RBS             477..499
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    514..538
                     /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     complement(538..557)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(539..557)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(645..664)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        870..947
                     /gene="lacI"
                     /label=lacI promoter
     CDS             948..2030
                     /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(967..986)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2839..3030
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3024..3046)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3132..3271
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3187..3206
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3286..3303)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3457..4045)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3537..3556)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4156..4175)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4167..4982
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4167..4982
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4240..4259)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5074..5529)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5211..5232)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5423..5442
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagagaaatt taaccacgag
      181 tttattaaga gtgtgcaccg ccttattatt tttcaaactg cggatgagac caaccgccag
      241 tacccagttt tttgctatcc tgtttcagca cgcacggggt gcgagagctg ctcagcgggg
      301 tcaggcgcgc gctgctcgcc atgctcaggg ttttcgccag gctgcgcgct ttccacaggc
      361 tcgcatgctg tttatacatg ctcggaataa acataatcag caggctcggc agaagcaggc
      421 taagcagaag cagaaacgcc agaatatcca gtaaggtcca gcgttccata tgtatatctc
      481 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
      541 tatagtgagt cgtattaatt tcgcgggatc gagatcgatc tcgatcctct acgccggacg
      601 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
      661 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
      721 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
      781 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
      841 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg
      901 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
      961 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     1021 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     1081 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     1141 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     1201 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     1261 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     1321 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     1381 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     1441 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     1501 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     1561 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     1621 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     1681 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     1741 gtgcggacat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     1801 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     1861 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     1921 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     1981 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     2041 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc
     2101 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     2161 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
     2221 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
     2281 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
     2341 aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg
     2401 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     2461 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     2521 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     2581 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     2641 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     2701 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     2761 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     2821 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     2881 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     2941 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
     3001 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     3061 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     3121 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     3181 actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca
     3241 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     3301 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3361 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3421 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3481 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3541 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3601 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3661 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3721 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3781 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3841 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     3901 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3961 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4021 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4081 tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt
     4141 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta
     4201 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     4261 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     4321 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     4381 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     4441 atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag
     4501 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     4561 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg
     4621 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     4681 gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt
     4741 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     4801 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     4861 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     4921 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
     4981 aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5041 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     5101 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     5161 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     5221 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     5281 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     5341 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     5401 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     5461 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     5521 agggcgcgtc ccattcgcca
//
