LOCUS       sequence-311159-        3240 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 3240)
  AUTHORS   Shin J, Noireaux V
  TITLE     Efficient cell-free expression with the endogenous E. Coli RNA 
            polymerase and sigma factor 70.
  JOURNAL   J Biol Eng. 2010 Jun 24;4:8.
  PUBMED    20576148
REFERENCE   2  (bases 1 to 3240)
  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: "J Biol 
            Eng."; date: "2010-06-24"; volume: "4"; pages: "8"
COMMENT     Sequence Label: pBEST-p15A-PLtetO1-tetR-T500
FEATURES             Location/Qualifiers
     source          1..3240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             68..90
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             97..720
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(170..189)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     rep_origin      1007..1552
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     1453..1472
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1669..1686
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(1919..2710,2711..2779))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2542..2561
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2780..2884)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2952..2970
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacagctag
       61 caataatttt gtttaacttt aagaaggaga tataccatgt ctagattaga taaaagtaaa
      121 gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt
      181 aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt aaaaaataag
      241 cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac tcacttttgc
      301 cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag ttttagatgt
      361 gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc tacagaaaaa
      421 cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt ttcactagag
      481 aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt attggaagat
      541 caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag tatgccgcca
      601 ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc cttcttattc
      661 ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag tgggtcttaa
      721 ctcgagcaaa gcccgccgaa aggcgggctt ttctgtgtcg accgatgccc ttgagagcct
      781 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
      841 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc atgataagct gtcaaacatg
      901 agaattgcaa cttatatcgt atggggctga cttcaggtgc tacatttgaa gagataaatt
      961 gcactgaaat ctagaaatat tttatctgat taataagatg atcttcttga gatcgttttg
     1021 gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc ggtttttcga
     1081 aggttctctg agctaccaac tctttgaacc gaggtaactg gcttggagga gcgcagtcac
     1141 caaaacttgt cctttcagtt tagccttaac cggcgcatga cttcaagact aactcctcta
     1201 aatcaattac cagtggctgc tgccagtggt gcttttgcat gtctttccgg gttggactca
     1261 agacgatagt taccggataa ggcgcagcgg tcggactgaa cggggggttc gtgcatacag
     1321 tccagcttgg agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat gagacaaacg
     1381 cggccataac agcggaatga caccggtaaa ccgaaaggca ggaacaggag agcgcacgag
     1441 ggagccgcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccaccactg
     1501 atttgagcgt cagatttcgt gatgcttgtc aggggggcgg agcctatgga aaaacggctt
     1561 tgccgcggcc ctctcacttc cctgttaagt atcttcctgg catcttccag gaaatctccg
     1621 ccccgttcgt aagccatttc cgctcgccgc agtcgaacga ccgagcgtag cgagtcagtg
     1681 agcgaggaag cggaatatat cctgtatcac atattctgct gacgcaccgg tgcagccttt
     1741 tttctcctgc cacatgaagc acttcactga caccctcatc agtgccaaca tagtaagcca
     1801 gtatacactc cgctagggtc atgagattat caaaaaggat cttcacctag atccttttaa
     1861 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     1921 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     1981 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2041 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2101 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2161 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2221 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2281 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2341 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2401 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2461 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     2521 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     2581 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2641 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2701 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2761 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     2821 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     2881 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     2941 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc tggcgaatcc
     3001 tctgaccagc cagaaaacga cctttctgtg gtgaaaccgg atgctgcaat tcagagcgcc
     3061 agcaagtggg ggacagcaga agacctgacc gccgcagagt ggatgtttga catggtgaag
     3121 actatcgcac catcagccag aaaaccgaat tttgctgggt gggctaacga tatccgcctg
     3181 atgcgtgaac gtgacggacg taaccaccgc gacatgtgtg tgctgttccg ctgggcatgc
//
