LOCUS       sequence-414414-        4568 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ColE1 ori, KanR, TetR, Tet promoter with a codon optimized pbTTA 
            gene bearing an N-terminal hexahistidine tag followed by a SUMO tag 
            and a TEV protease cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4568)
  AUTHORS   Jones MA, Butler ND, Anderson SR, Wirt SA, Govil I, Lyu X, Fang Y, 
            Kunjapur AM
  TITLE     Discovery of L-threonine transaldolases for enhanced biosynthesis of
            beta-hydroxylated amino acids.
  JOURNAL   Commun Biol. 2023 Sep 11;6(1):929. doi: 10.1038/s42003-023-05293-0.
  PUBMED    37696954
REFERENCE   2  (bases 1 to 4568)
  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: "Commun 
            Biol."; date: "2023-09-11"; volume: "6(1):929. doi"; pages: " 
            10.1038/s42003-023-05293-0"
COMMENT     Sequence Label: P25-pZE-SUMO-PbTTA
FEATURES             Location/Qualifiers
     source          1..4568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     195..214
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     rep_origin      complement(534..1122)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(614..633)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1210..1304)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1335..2129)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRQ
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1446..1465)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2056..2075
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2308..2326
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2347..2365,2366..2371,2372..2388,2389..2394,
                     2395..2420)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2347..2365
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2372..2390
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2430..2441
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2460..2477
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2808..2828
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      4139..4185
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(4232..4568,1..290))
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
ORIGIN
        1 ccatcgcgat gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct
       61 tgccagcttt ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg
      121 gctaaggcgt cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct
      181 acacctagct tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc
      241 agctctaatg cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa
      301 ttgctagcat tatacctagg actgaactag ccataaagtt gacactacgc tctcctgagt
      361 aggacaaatc cgccgcccta gacctagggc tttcggctgc ggcgagcggt atcagctcac
      421 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      481 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      541 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      601 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      661 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      721 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      781 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      841 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      901 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      961 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1021 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1081 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1141 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgact
     1201 agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc
     1261 cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg agctctcgaa
     1321 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1381 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1441 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1501 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     1561 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     1621 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     1681 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     1741 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     1801 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     1861 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     1921 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     1981 ttgacaaaaa gaacctggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     2041 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2101 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2161 gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg
     2221 cagggcttcc caaccttacc agagggcgcc ccagctggca attccgacgt ctaagaaacc
     2281 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac
     2341 ctcgagtccc tatcagtgat agagattgac atccctatca gtgatagaga tactgagcac
     2401 atcagcagga cgcactgacc gaattcatta aagaggagaa aggtaccatg ggcagcagcc
     2461 atcaccatca tcaccacatg tccctgcagg actcggaggt taaccaggaa gcaaagccgg
     2521 aagtcaaacc ggaagtgaaa cccgaaactc acatcaatct gaaggtaagt gatggttctt
     2581 cagagatatt ctttaaaatt aaaaaaacca cgcctctgcg gcgtcttatg gaagcgttcg
     2641 ccaaacgaca agggaaagag atggatagct tacgttttct ctatgatggc attcgcatcc
     2701 aggcggatca agctccagag gacttggata tggaagataa cgacattatc gaagcccatc
     2761 gcgaacagat tggtggcggc ggtggtgggt cgggaggtgg aggttcagaa aatctgtatt
     2821 ttcagggcga aacctccctg aaggattttg aaactatcct tcacttaatt aataaggagg
     2881 agattgactc aaatgacacc attcatatga ccgccaacga aaatattatg tctaaattgt
     2941 ccaaacacta cttaaaaagc actttgtctt accgctacca tgtcggaatg ttcgatgatc
     3001 aaaagaacct gacagtctcg cgttcgtgtc ttatcaaaaa ctctttgatg ctgcgttgcc
     3061 tttcacccat cttcctgtta gaacaacaag cccgtgaata cgtaaaaaaa atgttcttcg
     3121 ctgagtatgc ggactttcgt cctttgtccg gtatgcacac cgttttttgt atcttatcta
     3181 ccttaacaaa accgaacgat cgtgtctatg tcttcacgac cgaatcggta ggacacgcag
     3241 ccacagtttc tttattgaag tcgttgggtc gcaaagtgtc cttcatccca ttttgtgaga
     3301 agaaacttga tattgactta gagaagctga gtaaacaaat cttgattgag aaacccaacg
     3361 caattctttt tgattttggt actccattct acccattgcc gatccgcgaa attcgcgaga
     3421 ttgtaggaaa cgacgtgaag atgatttatg acgcctcgca tgtgttgggt ttgattgcgg
     3481 gtggacagtt ccaaaatcca cttcttgaag gctgtgacgt gctgatcgga aatactcaca
     3541 agacatttcc ggggccgcag aaaggcatga tcttgtataa aaacaagtct ttgggaaagg
     3601 agatcgcaac agaaattttc aaatcagcca tttctgcgca gcatactcat catgctatcg
     3661 ccctgtacgt tactatcatt gaaatgtata tccacgggaa ggaatacgcc aaccaaatca
     3721 tcaaaaataa tcatgcgtta tcccaggcat taatcaatga aggttttaaa atttttaagc
     3781 gtaaaaacca gtttagcctt agtcacatga ttgcgattac gggggatttt ccgattgatc
     3841 atcatgttgc atgtgccgat ttgcataatt ctaacatctc cacaaattcg cgtattctgt
     3901 atgactttcc agccgtgcgc attggcgttc aggaggttac acgtaaagga atgaaagaaa
     3961 aggatatggt gcaattagcc aaatttttta aggaaatcat cctggatcgc aagaacatca
     4021 gctctaaaat caaggagttc aataacaaat tcaatagtat tgaatatagt cttgacgaga
     4081 tctacgagaa gttattctaa cttgatgggg gatcccatgg tacgcgtgct agaggcatca
     4141 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     4201 gaacgtctca ttttcgccag atatcgacgt cttaagaccc actttcacat ttaagttgtt
     4261 tttctaatcc gcatatgatc aattcaaggc cgaataagaa ggctggctct gcaccttggt
     4321 gatcaaataa ttcgatagct tgtcgtaata atggcggcat actatcagta gtaggtgttt
     4381 ccctttcttc tttagcgact tgatgctctt gatcttccaa tacgcaacct aaagtaaaat
     4441 gccccacagc gctgagtgca tataatgcat tctctagtga aaaaccttgt tggcataaaa
     4501 aggctaattg attttcgaga gtttcatact gtttttctgt aggccgtgta cctaaatgta
     4561 cttttgct
//
