LOCUS       sequence-6888-ex        7098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7098)
  AUTHORS   Milne JC, Blanke SR, Hanna PC, Collier RJ
  TITLE     Protective antigen-binding domain of anthrax lethal factor mediates 
            translocation of a heterologous protein fused to its amino- or 
            carboxy-terminus.
  JOURNAL   Mol Microbiol. 1995 Feb . 15(4):661-6.
  PUBMED    7783638
REFERENCE   2  (bases 1 to 7098)
  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: "Mol 
            Microbiol. 1995 Feb . 15(4):661-6."
COMMENT     Sequence Label: pET-15b LFN-DTA
FEATURES             Location/Qualifiers
     source          1..7098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     130..149
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        130..148
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    149..173
                     /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)."
     RBS             188..210
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             229..246
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             256..273
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1060..1077
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1725..1743)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1739..1786
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1961..1966,1967..1983,1984..1989))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2016..2034)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2102..2206
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2207..2275,2276..3067)
                     /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     complement(2425..2444)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3238..3826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3727..3746
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3980..3997
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4012..4154
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4080..4099)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4240..4262
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4256..4447)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5759..6841)
                     /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     6803..6822
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6842..6919)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
       61 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      121 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
      181 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
      241 catcacagca gcggcctggt gccgcgcggc agccatatgg cgggcggtca tggtgatgta
      301 ggtatgcacg taaaagagaa agagaaaaat aaagatgaga ataagagaaa agatgaagaa
      361 cgaaataaaa cacaggaaga gcatttaaag gaaatcatga aacacattgt aaaaatagaa
      421 gtaaaagggg aggaagctgt taaaaaagag gcagcagaaa agctacttga gaaagtacca
      481 tctgatgttt tagagatgta taaagcaatt ggaggaaaga tatatattgt ggatggtgat
      541 attacaaaac atatatcttt agaagcatta tctgaagata agaaaaaaat aaaagacatt
      601 tatgggaaag atgctttatt acatgaacat tatgtatatg caaaagaagg atatgaaccc
      661 gtacttgtaa tccaatcttc ggaagattat gtagaaaata ctgaaaaggc actgaacgtt
      721 tattatgaaa taggtaagat attatcaagg gatattttaa gtaaaattaa tcaaccatat
      781 cagaaatttt tagatgtatt aaataccatt aaaaatgcat ctgattcaga tggacaagat
      841 cttttattta ctaatcagct taaggaacat cccacagact tttctgtaga attcttggaa
      901 caaaatagca atgaggtaca agaagtattt gcgaaagctt ttgcatatta tatcgagcca
      961 cagcatcgtg atgttttaca gctttatgca ccggaagctt ttaattacat ggataaattt
     1021 aacgaacaag aaataaatct atccgccatg ggcagctctc accaccacca ccaccactct
     1081 tccggcctgg ttccacgtgg tgctgacgac gttgttgact cttctaaatc tttcgttatg
     1141 gaaaacttct cttcttacca cggtaccaaa ccgggttacg tcgactctat ccagaaaggt
     1201 atccagaagc cgaaatctgg tacccagggt aactacgacg acgactggaa aggtttctac
     1261 tctaccgaca acaaatacga cgccgcgggt tactctgttg acaacgaaaa cccgctgtct
     1321 ggtaaagctg gtggtgttgt taaagttacc tacccgggtc tgaccaaagt tctggctctg
     1381 aaagttgaca acgctgaaac catcaaaaaa gaactgggtc tctctctgac cgaaccgctg
     1441 atggaacagg ttggtaccga agaattcatc aaacgtttcg gtgacggtgc ttctcgtgtt
     1501 gttctgtctc tgccgttcgc tgagggctct tcttctgttg aatacatcaa caactgggaa
     1561 caggctaaag ctctgtctgt tgaactggaa atcaacttcg aaacccgtgg taaacgtggc
     1621 caggacgcta tgtacgaata catggctcag gcttgtgcag gtaaccgtta atgaggatcc
     1681 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1741 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1801 tatatccgga tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca
     1861 gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt
     1921 gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata
     1981 agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     2041 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     2101 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2161 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2221 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2281 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2341 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     2401 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     2461 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     2521 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     2581 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     2641 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     2701 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     2761 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     2821 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     2881 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     2941 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3001 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3061 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3121 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3181 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3241 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3301 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3361 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3421 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3481 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3541 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3601 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3661 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3721 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3781 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3841 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3901 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3961 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4021 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt
     4081 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4141 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4201 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4261 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4321 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4381 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4441 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4501 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4561 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4621 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4681 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4741 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4801 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4861 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     4921 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     4981 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     5041 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     5101 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     5161 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     5221 cggtccagtg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     5281 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     5341 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     5401 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     5461 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     5521 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     5581 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     5641 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5701 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5761 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5821 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5881 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5941 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     6001 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     6061 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     6121 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     6181 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     6241 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6301 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6361 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6421 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6481 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6541 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6601 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6661 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6721 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6781 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6841 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     6901 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     6961 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     7021 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7081 caagcgctca tgagcccg
//
