LOCUS       sequence-372941-        6092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The reporter plasmid p11-LacY-wtx1 encodes the toxin CcdB gene under
            the inducible BAD promoter and a single copy of the endonuclease 
            cleavage site which is readily extendable to multiple copies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6092)
  AUTHORS   Chen Z, Zhao H
  TITLE     A highly sensitive selection method for directed evolution of homing
            endonucleases.
  JOURNAL   Nucleic Acids Res. 2005 Oct 6;33(18):e154.
  PUBMED    16214805
REFERENCE   2  (bases 1 to 6092)
  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: "Nucleic 
            Acids Res."; date: "2005-10-6"; volume: "33(18)"; pages: "e154"
COMMENT     Sequence Label: p11-LacY-wtx1
FEATURES             Location/Qualifiers
     source          1..6092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(485..940)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(622..643)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     834..853
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(join(982..1773,1774..1842))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1605..1624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1843..1934)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(1953..1980)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2072..2158)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2311..2328
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(2423..2728)
                     /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     complement(2525..2544)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        complement(2760..3044)
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     complement(2818..2837)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             3071..3949
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     primer_bind     5396..5412
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5428..5444)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5428..5444)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5441..5463)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5452..5468
                     /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        complement(join(5476..5482,5483..5500,5501..5506))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5521..5542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5707..5724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5878..6092,1..374))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5958..5977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
       61 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      121 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      181 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
      241 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      301 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      361 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      421 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
      481 ttttaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
      541 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagccc
      601 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
      661 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
      721 cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
      781 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
      841 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
      901 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt aaatcaatct aaagtatata
      961 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     1021 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     1081 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     1141 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     1201 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     1261 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     1321 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     1381 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     1441 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     1501 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     1561 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca
     1621 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     1681 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     1741 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     1801 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1861 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1921 gaaaaataaa caaaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct
     1981 gcttaatttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt
     2041 gcttcgcaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg
     2101 acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat
     2161 gcctggcagt tccctactct cgcatgggga gaccccacac taccatcggc gctacggcgt
     2221 ttcacttctg agttcggcat ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa
     2281 ttctgtttta tcagaccgct tctgcgttct gatttaatct gtatcaggct gaaaatcttc
     2341 tctcatccgc caaaacagcc aagcttgcat gccgtatgtc tagagcgtga tattaccctg
     2401 ttatccctag cgtaatgcta gattatattc cccaaaacat caggttaatg gcgtttttga
     2461 tgtcattttc gcggtggctg agatcagcca cttcttcccc gataacggag accggcacac
     2521 tggccatatc ggtggtcatc atgcgccagc tttcatcccc gatatgcacc accgggtaaa
     2581 gttcacggga gactttatct gacagcagac gtgcactggc cagggggatc accatccgtc
     2641 gcccgggcgt gtcaataata tcactctgta catccacaaa cagacgataa cggctctctc
     2701 ttttataggt gtaaacctta aactgcatcg ttttcaatcg ctagcccaaa aaaacgggta
     2761 tggagaaaca gtagagagtt gcgataaaaa gcgtcaggta ggatccgcta atcttatgga
     2821 taaaaatgct atggcatagc aaagtgtgac gccgtgcaaa taatcaatgt ggacttttct
     2881 gccgtgatta tagacacttt tgttacgcgt ttttgtcatg gctttggtcc cgctttgtta
     2941 cagaatgctt ttaataagcg gggttaccgg tttggttagc gagaagagcc agtaaaagac
     3001 gcagtgacgg caatgtctga tgcaatatgg acaattggtt tcttctctga atggcgggag
     3061 tatgaaaagt atggctgaag cgcaaaatga tcccctgctg ccgggatact cgtttaatgc
     3121 ccatctggtg gcgggtttaa cgccgattga ggccaacggt tatctcgatt tttttatcga
     3181 ccgaccgctg ggaatgaaag gttatattct caatctcacc attcgcggtc agggggtggt
     3241 gaaaaatcag ggacgagaat ttgtttgccg accgggtgat attttgctgt tcccgccagg
     3301 agagattcat cactacggtc gtcatccgga ggctcgcgaa tggtatcacc agtgggttta
     3361 ctttcgtccg cgcgcctact ggcatgaatg gcttaactgg ccgtcaatat ttgccaatac
     3421 ggggttcttt cgcccggatg aagcgcacca gccgcatttc agcgacctgt ttgggcaaat
     3481 cattaacgcc gggcaagggg aagggcgcta ttcggagctg ctggcgataa atctgcttga
     3541 gcaattgtta ctgcggcgca tggaagcgat taacgagtcg ctccatccac cgatggataa
     3601 tcgggtacgc gaggcttgtc agtacatcag cgatcacctg gcagacagca attttgatat
     3661 cgccagcgtc gcacagcatg tttgcttgtc gccgtcgcgt ctgtcacatc ttttccgcca
     3721 gcagttaggg attagcgtct taagctggcg cgaggaccaa cgtatcagcc aggcgaagct
     3781 gcttttgagc accacccgga tgcctatcgc caccgtcggt cgcaatgttg gttttgacga
     3841 tcaactctat ttctcgcggg tatttaaaaa atgcaccggg gccagcccga gcgagttccg
     3901 tgccggttgt gaagaaaaag tgaatgatgt agccgtcaag ttgtcataat tggtaacgaa
     3961 tcagacaatt gacggcttga cggagtagca tagggtttgc agaatccctg cttcgtccat
     4021 ttgacaggca cattatgcat cgaggccttt aagcgacttc attcacctga cgacgcagca
     4081 gggaaagcgg gcccgggccg ctaagcgtga acacggaaat taaggtgaag cccagcgcca
     4141 ccagacccag caccagataa gcgccttgga aacctatgct ttcatacata ttgcccgcca
     4201 gtacagacat aaaaatcatc gccagttgct taaagaagct gaaactgacc agataaatcg
     4261 tcgctgaaaa acgcacttca aactgactag taatatattt aaaggagccc accagcagga
     4321 acggtacttc aaacatatgc agcgttttca gaataaccac ttccagcgct gaggtggcga
     4381 acgatgagcc aataatacgt acagacataa tagtgccagc cagcagcagg gcattcttcc
     4441 caccgatgcg attaatgatc agtggcgcaa agaacataat cgaggcgtta agtaattcgc
     4501 ccattgtcgt tacgtagcca aatacgcggg taccctgttc accggtagca aagaacgaag
     4561 taaagaaatt agcaaactgt tggtcaaaaa catcgtaggt ggaggaaacg ccaataacat
     4621 acagtgacaa aaaccacagt tttggctgtc tgaacagttc cagtgccagc ttaaggctaa
     4681 atgccgaatg gttggcacct accgcattgg caaccgtggc actcgagggc gcgtccgttt
     4741 tggcgaaaaa gagtaaaacg gcgaggatga gacaactgcc agagcccagc cagaaaacaa
     4801 actgattatt aatggtgaac atgatcccga caatcgaggc aaccagcgcc cagccaacac
     4861 tgccaaacat ccgcgcgcga ccaaattcga aattactgcg ccggctgact ttctcaataa
     4921 atgcctctac tgctggcgca ccggcgttaa aactaaagcc tagataaata ccaccaacaa
     4981 tcgatcctac taaaatgttg tactgcagca gtggcccgaa gataaaaata aagaacggcg
     5041 caaacatcac taacatgccg gtaataatcc acagcaggta tttgcgtaga ccgagtttgt
     5101 cagaaagcag accaaacagc ggttggaata atagcgagaa cagagaaata gcggcaaaaa
     5161 taatacccgt atcacttttg ctgatatggt tgatatcatg tagccaaatc gggaaaaacg
     5221 ggaagtaggc tcccatgata aaaaagtaaa agaaaaagaa taaaccgaac atccaaaagt
     5281 ttgtgttttt caaatagtac atggatcctt tccttattac gcctgctggc gaaaggggga
     5341 tgtgctgcaa ggcgattaag ttgggtaacg cccgggtttt accagtcacg acgttgtaaa
     5401 acgacggcca gtgaatccgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5461 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagccc ggggtcccta
     5521 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5581 cctgtcgtgc cagggaattc cggtctccct atagtgagtc aggcctattt aaatcgtacg
     5641 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     5701 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5761 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5821 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5881 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5941 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6001 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6061 ggcgctttct catagctcac gctgtaggta tc
//
