LOCUS       sequence-414417-        4628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ColE1 ori, KanR, TetR, Tet promoter with a codon optimized piTTA 
            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 4628)
  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 4628)
  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: P15-pZE-SUMO-PiTTA
FEATURES             Location/Qualifiers
     source          1..4628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(59..105)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1476..1496)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1827..1844)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1863..1874
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(join(1884..1909,1910..1915,1916..1932,1933..1938,
                     1939..1957))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1914..1932
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1939..1957
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(1978..1996)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             2175..2969
                     /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(2229..2248)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2839..2858
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      3000..3094
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      3182..3770
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3671..3690
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3967..4001
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     CDS             join(4014..4628,1..12)
                     /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(4090..4109)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 agtgggtctt aagacgtcga tatctggcga aaatgagacg ttcaccgaca aacaacagat
       61 aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tctagcacgc
      121 gtaccatggg atcccccatc aagttacgtt acttattagc gttgaattcc acgctcaagc
      181 aaacggcggc gagactcgtc gatttgttga tcgaacgagt aggccagagg ggcaagtgga
      241 aaatcgccca ataactcctt cacctttgaa gcaacacgtg caggttcggt acgcttgaag
      301 taaaggtcag cgataaggcc agcgacgcga cgaaagtcat cctctttcat accgcggcgg
      361 gtcatggcct gtacccccaa gcggatcccg tgcatcccgg gctttcccgg gatctcgatg
      421 ttagtcgagc ggattccgcc cgcgtgtaac gagtccacgc acatactcaa cgcctgttgt
      481 aaatctccta cggcgaaatg cacctggtgc gtctctgtaa atccaaaaga ctctcccgaa
      541 acatttaaac caagctcgtg aagttggttg gccaacgcct tagcattgcg caccatctgg
      601 tgggcatagg cggggccaaa agcttccatc tccttgaatg cggcccacat agagggtaaa
      661 agttcggcgt gacagttact ctgtaagtgt ggacataccc aaagactggt ttccaccaac
      721 agggggtgct gagcgctctt gaacgcaata aacccctttt gaggccccgg aatagtcttg
      781 tgagtattgc cgtgagcgat atccgcaccg cacgccaatg ggctatcgaa gaccccaccc
      841 agaataaggc ccccatcatg agacatatca taagttaaag tgcatgaatc gggaaggatt
      901 gcacgaatct cggctaacgg ctgccaacga agtttgaagg attgatcaag aattacgata
      961 cggatgtgag ggttacggcg gaccatgtca tcaagcttag caacgtcaat aagcagactt
     1021 tgcggatcca caggtaaatg gaaaatctca atacccattt ttgtcgccaa ttgctctaaa
     1081 gcaaaatgcc ccccatcgcg atgtgcaaag tgtacaaaac cttcaccttg tttacaagcc
     1141 gctaacatca aggcctgttc agccgggctc ccaccattag ggcgccaatc gaatgcctgc
     1201 gcacccatca tacgtttggc caaaccgcgc agatcatcgg cgatctcgtt catttgtcct
     1261 ggctctggga aatgccattc tccaagagga acggggaacg gaaaagagca gtgataaaat
     1321 gccccggctg tagagccgct ggtcagacga acaagctctg aagggtagtt ttcgttagct
     1381 gtaagtgaaa gtgtgtcctg gcggtacttg tagtcctgtc caagggtctg agccagccag
     1441 tcaatgacag gaccaacatt cgattcgtct tgtttgccct gaaaatacag attttctgaa
     1501 cctccacctc ccgacccacc accgccgcca ccaatctgtt cgcgatgggc ttcgataatg
     1561 tcgttatctt ccatatccaa gtcctctgga gcttgatccg cctggatgcg aatgccatca
     1621 tagagaaaac gtaagctatc catctctttc ccttgtcgtt tggcgaacgc ttccataaga
     1681 cgccgcagag gcgtggtttt tttaatttta aagaatatct ctgaagaacc atcacttacc
     1741 ttcagattga tgtgagtttc gggtttcact tccggtttga cttccggctt tgcttcctgg
     1801 ttaacctccg agtcctgcag ggacatgtgg tgatgatggt gatggctgct gcccatggta
     1861 cctttctcct ctttaatgaa ttcggtcagt gcgtcctgct gatgtgctca gtatctctat
     1921 cactgatagg gatgtcaatc tctatcactg atagggactc gaggtgaaga cgaaagggcc
     1981 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcgg
     2041 aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg
     2101 ctttcttgcc gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat
     2161 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     2221 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     2281 tgttccggct gtcagcgcag gggcgccagg ttctttttgt caagaccgac ctgtccggtg
     2341 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     2401 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     2461 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     2521 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     2581 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     2641 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     2701 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     2761 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     2821 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     2881 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     2941 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaga gctcgcttgg
     3001 actcctgttg atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct
     3061 cggttgccgc cgggcgtttt ttattggtga gaatccaagc actagtcatg accaaaatcc
     3121 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3181 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3241 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3301 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3361 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3421 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3481 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3541 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3601 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3661 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3721 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3781 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3841 cgttatccac agaatcaggg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3901 gccgcagccg aaagccctag gtctagggcg gcggatttgt cctactcagg agagcgtagt
     3961 gtcaacttta tggctagctc agtcctaggt acaatgctag caattaggaa ttaatgatgt
     4021 ctagattaga taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa
     4081 tcgaaggttt aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt
     4141 attggcatgt aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata
     4201 ggcaccatac tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata
     4261 acgctaaaag ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag
     4321 gtacacggcc tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc
     4381 aacaaggttt ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt
     4441 taggttgcgt attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta
     4501 ctactgatag tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg
     4561 cagagccagc cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta
     4621 aatgtgaa
//
