LOCUS       sequence-414415-        4670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ColE1 ori, KanR, TetR, Tet promoter with a codon optimized buTTA 
            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 4670)
  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 4670)
  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: P18-pZE-SUMO-BuTTA
FEATURES             Location/Qualifiers
     source          1..4670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(329..349)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(680..697)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             716..727
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(join(737..762,763..768,769..785,786..791,
                     792..810))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    767..785
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    792..810
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(831..849)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1028..1822
                     /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(1082..1101)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1692..1711
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1853..1947
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2035..2623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2524..2543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             2867..3493
                     /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     complement(2943..2962)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      complement(3540..3586)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tagcctcgaa caactgtttt cccaaactag caagtttctc gaccagactc gactgcgcgc
       61 cactgtcagg gaagtaccac tctcctgggt tagcggcgta aggtggcgcg aattcataga
      121 aaccgccttg cataccggct cccagcgtgc gcacaaagcg ggatggataa ttctcagagg
      181 cggtcagact taagtggctt gcgcggtata aattatccaa ggcaaaatcg ctatgaagac
      241 tatcgaagat gtttgagatt ttttctacta aacgggactt acgctgctca tctacgaacg
      301 ggtttactac cgcctgtgcg aagtccgtgc cctgaaaata cagattttct gaacctccac
      361 ctcccgaccc accaccgccg ccaccaatct gttcgcgatg ggcttcgata atgtcgttat
      421 cttccatatc caagtcctct ggagcttgat ccgcctggat gcgaatgcca tcatagagaa
      481 aacgtaagct atccatctct ttcccttgtc gtttggcgaa cgcttccata agacgccgca
      541 gaggcgtggt ttttttaatt ttaaagaata tctctgaaga accatcactt accttcagat
      601 tgatgtgagt ttcgggtttc acttccggtt tgacttccgg ctttgcttcc tggttaacct
      661 ccgagtcctg cagggacatg tggtgatgat ggtgatggct gctgcccatg gtacctttct
      721 cctctttaat gaattcggtc agtgcgtcct gctgatgtgc tcagtatctc tatcactgat
      781 agggatgtca atctctatca ctgataggga ctcgaggtga agacgaaagg gcctcgtgat
      841 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt cggaattgcc
      901 agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt
      961 gccgccaagg atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc
     1021 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     1081 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     1141 gctgtcagcg caggggcgcc aggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     1201 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     1261 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     1321 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     1381 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     1441 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     1501 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     1561 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     1621 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     1681 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     1741 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     1801 ccttcttgac gagttcttct gagcgggact ctggggttcg agagctcgct tggactcctg
     1861 ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc
     1921 cgccgggcgt tttttattgg tgagaatcca agcactagtc atgaccaaaa tcccttaacg
     1981 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2041 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2101 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2161 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2221 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2281 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2341 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2401 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2461 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2521 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2581 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2641 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2701 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2761 ccgaaagccc taggtctagg gcggcggatt tgtcctactc aggagagcgt agtgtcaact
     2821 ttatggctag ttcagtccta ggtataatgc tagcaattag gaattaatga tgtctagatt
     2881 agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg
     2941 tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca
     3001 tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca
     3061 tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa
     3121 aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg
     3181 gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg
     3241 tttttcacta gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg
     3301 cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga
     3361 tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc
     3421 agccttctta ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga
     3481 aagtgggtct taagacgtcg atatctggcg aaaatgagac gttcaccgac aaacaacaga
     3541 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctctagcacg
     3601 cgtaccatgg gatcccccat caagttatta agcgattact tcctccatca atttaatacc
     3661 cgaaggggta taatagtgag catctaatga ataatgaagc gtattaatag gatatgattc
     3721 aagaaatgac gcaatttcgt tgcggatggc cgtgggggat tcgtttttca gaatcaagcg
     3781 cactaaaaaa cgggcaactt cagccatccc gtgctcttta attccgcgac gcgtcaatgc
     3841 ctgcactcct aagcgcaaac caaataagcc gttcgcgcct ggaatctcga tgttgttgca
     3901 gcgaattcct gccaatgcca aggtaccagt aactaactcc aacgctttac gctcggaccc
     3961 aacaacaaca tgaacttggt gtgtttctgt aaaaccgaac gattcgcccg agacgcgtac
     4021 tccctcggcg tgcagggcgt gtgccaacgc cttagcattt ttaataacct gctccgcgta
     4081 actgcgtccg tacaaagaca tttcaaccaa ggcaatatac atgggtgcga tcaactcggc
     4141 atgcgagtta gattgcaaat gaggacaaac ccaatcgcta acatgcttca tcgcaggatg
     4201 gtctacattc ttgaacgcaa tgtaaccctt ttgcgggccc ggaattgtct tgtgtgtatt
     4261 cccgtgaaca atatccgctc cgaaaagtaa tggctggggc aggcattcgc cgatgattaa
     4321 tcccccgtcg tgactcgcgt cgtacgataa tacgactgat tccggtaagg cttggcggat
     4381 ttgcagtaaa ggttgccagc gaagcttaaa cgattggtcc agaattacaa gcttaatgtg
     4441 ggggttatct ttgattaatg tcgccaggcg gtcaacatca atcaaaagac tcttctcctc
     4501 gattggcaga tggaagatgc taactcccac cttctgggcc aactcttcca gagcaaagtg
     4561 gccgccatcc ttgtgagcaa agtggacgaa gccatccccg cgggcacatg tgcctaaaag
     4621 cacagcctgt tccgctgctg atcccccgtt gggacgccag tcaaacgagt
//
