LOCUS       sequence-327989-        3708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two valuable constructs for either IPTG-induced or optimized 
            temperature-induced expression can be simultaneously obtained in a 
            single cloning process by utilizing plasmid pANY4-pL18-ccdB..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3708)
  TITLE     Yingfeng An Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3708)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pANY4-pL18-ccdB
FEATURES             Location/Qualifiers
     source          1..3708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             87..104
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             126..146
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             509..814
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     693..712
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(879..896)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    974..988
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     CDS             1210..1923
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     primer_bind     complement(1937..1955)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1951..1998
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2066..2071,2072..2088,2089..2094))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             complement(2107..2922)
                     /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             complement(2107..2922)
                     /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     2830..2849
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2914..2933
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3044..3632
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3533..3552
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atgggccacc atcatcacca ccatgattat gatattccaa
      121 ctaccgagaa tttgtatttt caggggggat ccactagtag gcctgtcgac ggtcagtccg
      181 gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa
      241 tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatga agcagcgtat
      301 tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc
      361 tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg
      421 aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct
      481 tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga
      541 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg
      601 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
      661 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
      721 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
      781 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca
      841 gtcgactgac cgtcaggcct ccatgggagc tcctgcaggt gatggtgatg gtgatgcata
      901 tgtatatctc cttaattttt aaccaatgct tcgtttcgta tcacacaccc caaagccttc
      961 tgctttgaat gctgcccttc ttcagggctt aatttttaag agcgtcacct tcatggtggt
     1021 cagtgcgtcc tgctgatgtg ctcagtatca ccgctagtgg tatttctgtc aacaccgcca
     1081 gagataattt atcaccgcag atggttatct gtatgttttt tatatgaatt tattttttgc
     1141 aggggggcat tgtttggtag gtgagacacg gtgttagata tttatccctt gcggtgatag
     1201 atttaacgta tgagcacaaa aaagaaacca ttaacacaag agcagcttga ggacgcacgt
     1261 cgccttaaag caatttatga aaaaaagaaa aatgaacttg gcttatccca ggaatctgtc
     1321 gcagacaaga tggggatggg gcagtcaggc gttggtgctt tatttaatgg catcaatgca
     1381 ttaaatgctt ataacgccgc attgcttaca aaaattctca aagttagcgt tgaagaattt
     1441 agcccttcaa tcgccagaga aatctacgag atgtatgaag cggttagtat gcagccgtca
     1501 cttagaagtg agtatgagta ccctgttttt tctcatgttc aggcagggat gttctcacct
     1561 aagcttagaa cctttaccaa aggtgatgcg gagagatggg taagcacaac caaaaaagcc
     1621 agtgattctg cattctggct tgaggttgaa ggtaattcca tgaccgcacc aacaggctcc
     1681 aagccaagct ttcctgacgg aatgttaatt ctcgttgacc ctgagcaggc tgttgagcca
     1741 ggtgatttct gcatagccag acttgggggt gatgagttta ccttcaagaa actgatcagg
     1801 gatagcggtc aggtgttttt acaaccacta aacccacagt acccaatgat cccatgcaat
     1861 gagagttgtt ccgttgtggg gaaagttatc gctagtcagt ggcctgaaga gacgtttggc
     1921 tgattggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1981 gtcttgaggg gttttttgct gaaaggagga actatatccg gatatccctg actgcgttag
     2041 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga
     2101 gaattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     2161 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     2221 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     2281 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     2341 gacgactgaa tccggtgaga atggcaaaag tttgtgcatt tctttccaga cttgttcaac
     2401 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     2461 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     2521 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     2581 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     2641 tgcgtcatca ggagtacgga taaaatgctt gatggtcgga agcggcataa attccgtcag
     2701 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     2761 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     2821 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     2881 tcgcggcctc gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     2941 gtttatgtaa gcagacagtt ttattgttca tgacaaaaat cccttaacgt gagttttcgt
     3001 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3061 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3121 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3181 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3241 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3301 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3361 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3421 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3481 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3541 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3601 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3661 tcctggcctt ttgctggcct ttggtaccag atctcgatcc cgcgaaat
//
