LOCUS       sequence-414416-        4619 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ColE1 ori, KanR, TetR, Tet promoter with a codon optimized csTTA 
            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 4619)
  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 4619)
  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: P17-pZE-SUMO-CsTTA
FEATURES             Location/Qualifiers
     source          1..4619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      39..85
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(132..758)
                     /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"
     primer_bind     663..682
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     rep_origin      complement(1002..1590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1082..1101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1678..1772)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1803..2597)
                     /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(1914..1933)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2524..2543
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2776..2794
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2815..2833,2834..2839,2840..2856,2857..2862,
                     2863..2888)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2815..2833
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2840..2858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2898..2909
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2928..2945
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3276..3296
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 cttgatgggg gatcccatgg tacgcgtgct agaggcatca aataaaacga aaggctcagt
       61 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgtctca ttttcgccag
      121 atatcgacgt cttaagaccc actttcacat ttaagttgtt tttctaatcc gcatatgatc
      181 aattcaaggc cgaataagaa ggctggctct gcaccttggt gatcaaataa ttcgatagct
      241 tgtcgtaata atggcggcat actatcagta gtaggtgttt ccctttcttc tttagcgact
      301 tgatgctctt gatcttccaa tacgcaacct aaagtaaaat gccccacagc gctgagtgca
      361 tataatgcat tctctagtga aaaaccttgt tggcataaaa aggctaattg attttcgaga
      421 gtttcatact gtttttctgt aggccgtgta cctaaatgta cttttgctcc atcgcgatga
      481 cttagtaaag cacatctaaa acttttagcg ttattacgta aaaaatcttg ccagctttcc
      541 ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca tctcaatggc taaggcgtcg
      601 agcaaagccc gcttattttt tacatgccaa tacaatgtag gctgctctac acctagcttc
      661 tgggcgagtt tacgggttgt taaaccttcg attccgacct cattaagcag ctctaatgcg
      721 ctgttaatca ctttactttt atctaatcta gacatcatta attcctaatt gctagcatta
      781 tacctaggac tgaactagcc ataaagttga cactacgctc tcctgagtag gacaaatccg
      841 ccgccctaga cctagggctt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      901 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      961 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1021 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1081 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1141 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1201 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1261 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1321 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1381 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1441 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1501 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1561 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1621 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgactag tgcttggatt
     1681 ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc
     1741 tgaggtcatt actggatcta tcaacaggag tccaagcgag ctctcgaacc ccagagtccc
     1801 gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
     1861 ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
     1921 cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
     1981 aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
     2041 acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc
     2101 gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga
     2161 gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca
     2221 agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
     2281 tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
     2341 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
     2401 cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
     2461 acctggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
     2521 tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
     2581 ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc
     2641 ctgcgccatc agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca
     2701 accttaccag agggcgcccc agctggcaat tccgacgtct aagaaaccat tattatcatg
     2761 acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcacct cgagtcccta
     2821 tcagtgatag agattgacat ccctatcagt gatagagata ctgagcacat cagcaggacg
     2881 cactgaccga attcattaaa gaggagaaag gtaccatggg cagcagccat caccatcatc
     2941 accacatgtc cctgcaggac tcggaggtta accaggaagc aaagccggaa gtcaaaccgg
     3001 aagtgaaacc cgaaactcac atcaatctga aggtaagtga tggttcttca gagatattct
     3061 ttaaaattaa aaaaaccacg cctctgcggc gtcttatgga agcgttcgcc aaacgacaag
     3121 ggaaagagat ggatagctta cgttttctct atgatggcat tcgcatccag gcggatcaag
     3181 ctccagagga cttggatatg gaagataacg acattatcga agcccatcgc gaacagattg
     3241 gtggcggcgg tggtgggtcg ggaggtggag gttcagaaaa tctgtatttt cagggcacgc
     3301 gcacgacccc ccaggcacgt catgtcgtgg agcgcctgaa ttcagtttta ggacaagact
     3361 accgctatcg tgaggattgt ctgagcctta ccgcgaatga gaactatcct tccgcattag
     3421 tgcgcttagc ggggagtgcc acagctggag ccttctacca ctgtagcttt ccgtttgagg
     3481 tgccaccggg agaatggtat tttcctgaga gcggtcgtat gggggaactt gctcaacagc
     3541 tgaatgaatt aggtcgttcg ttattaggcg cgggtacatt cgattggcgc cccaacggtg
     3601 gctcgccagc ggagcaggca ttgatgttag cggcctgcaa gcacggtgaa gggatggtcc
     3661 attttgctca tcgtgacggt ggccactttg cgctggagaa tctggcgcaa aaagctggta
     3721 tcgacatctt tcatttgcct gtagatcccc agacgttgtt gatcgatgtt gcacaccttg
     3781 acgagcttgt ccgccgcaat cctcaaatcc gtattgtgat cttggaccag tcttttaagt
     3841 tacgctggca accccttgca gcgatccgca aggttcttcc cccatcgtgt acacttacct
     3901 atgacacctc tcatgatggt ggacttatta tgggaggagt ttttgattct cccttgcatt
     3961 gtggtgcaga cgtaattcat ggcaacacgc ataaaacagt gcccggaccg cagaaggggt
     4021 atatcgcctt caaatccgct gagcatcctt tgttggttga cacgagtctg tggctttgcc
     4081 cacatttgca gtctaactgt catgccgagc ttttgcctcc aatgtgggtg gcttttaaag
     4141 aaatggaggc tttcggacat gattacgccc ctcaagtggc ccgcaacgcg aaggctctgg
     4201 cgggtcattt acatcgttta ggattcgagg tttcaggcga ggctttcggt ttcactgaaa
     4261 cccaccaagt gcattttgcc gtaggagact tgcagcaagc gcttgatttg tgcatgaaca
     4321 ccttgcatcg tgggggcatc cgctctacga atattgaaat cccgggtaaa cccggcattc
     4381 agggtattcg cctgggcgtt caggctatga cccgtcgcgg tctgcgcgaa gatgattttg
     4441 agcaggtggc gcgttttatc gcggacttgc acttccgcaa agcagaccca gccggagtcg
     4501 cagcacaagt agcggaattt cttcgtgctt ttcctttggc accattacat tactcatttg
     4561 atcaggaact ggatcatgag ttattgcagt cccttattgg ggaggcgtta cgctaataa
//
