LOCUS       sequence-125453-        3734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Post cloned 4-piece from Isothermal/Golden Gate assembly (GGA), 
            using sequences from 2 (PltetO1-UTR1), 3 (tetR+linker from primer 
            extension), 21 (linker from primer extension + deGFP), and 105 
            (vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3734)
  AUTHORS   Sun ZZ, Yeung E, Hayes CA, Noireaux V, Murray RM
  TITLE     Linear DNA for rapid prototyping of synthetic biological circuits in
            an Escherichia coli based TX-TL cell-free system.
  JOURNAL   ACS Synth Biol. 2014 Jun 20;3(6):387-97. doi: 10.1021/sb400131a. 
            Epub 2013 Dec 4.
  PUBMED    24303785
REFERENCE   2  (bases 1 to 3734)
  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"; doi: "10.1021/sb400131a";
            journalName: "ACS Synth Biol"; date: "2014-06-20- 20"; volume: "3"; 
            issue: "6"; pages: "387-97"
COMMENT     Sequence Label: pBEST-Pl-tetO1-tetR-linker-deGFP-T500 (109p)
FEATURES             Location/Qualifiers
     source          1..3734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             7..29
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             38..658
                     /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(111..130)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             662..682
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(722..743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(983..1002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1330..1351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      complement(1620..2208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1700..1719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2379..3170,3171..3239))
                     /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     3002..3021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3240..3344)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3412..3430
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    3674..3692
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3699..3717
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aataattttg tttaacttta agaaggagat ataccacatg tctagattag ataaaagtaa
       61 agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg
      121 taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa
      181 gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg
      241 ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg
      301 tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa
      361 acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga
      421 gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg tattggaaga
      481 tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc
      541 attattacga caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt
      601 cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtctgg
      661 tgaaaacctg tacttccagt ctggtggtgc tatggagctt ttcactggcg ttgttcccat
      721 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      781 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      841 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      901 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      961 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1021 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1081 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1141 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1201 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1261 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1321 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatctaac tcgagcaaag
     1381 cccgccgaaa ggcgggcttt tctgtgtcga ccgatgccct tgagagcctt caacccagtc
     1441 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgcggtatca
     1501 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1561 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1621 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1681 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1741 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1801 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1861 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1921 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1981 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2041 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2101 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2161 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2221 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2281 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2341 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2401 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2461 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2521 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     2581 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     2641 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     2701 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     2761 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     2821 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     2881 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     2941 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3001 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3061 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3121 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3181 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3241 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3301 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3361 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     3421 atcacgaggc cctttcgtct tcaagaattc tggcgaatcc tctgaccagc cagaaaacga
     3481 cctttctgtg gtgaaaccgg atgctgcaat tcagagcggc agcaagtggg ggacagcaga
     3541 agacctgacc gccgcagagt ggatgtttga catggtgatg actatcgcac catcagctaa
     3601 cgatatccgc ctgatgcgtg aacgtgacgg acgtaaccac cgcgacatgt gtgtgctgtt
     3661 ccgctgggca tgctccctat cagtgataga gattgacatc cctatcagtg atagagatac
     3721 tgagcacagc tagc
//
