LOCUS       sequence-414413-        4475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ColE1 ori, KanR, TetR, Tet promoter with a codon optimized kaTTA 
            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 4475)
  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 4475)
  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: P24-pZE-SUMO-KaTTA
FEATURES             Location/Qualifiers
     source          1..4475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             175..195
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1506..2132)
                     /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
                     RPTEKQYETLEKQLAFLCQQGFSLENALYALSAVGYFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     2037..2056
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(2145..2179)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     rep_origin      complement(2376..2964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2456..2475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3052..3146)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3177..3971)
                     /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(3288..3307)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3898..3917
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4150..4168
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(4189..4207,4208..4213,4214..4230,4231..4236,
                     4237..4262)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    4189..4207
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4214..4232
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             4272..4283
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4302..4319
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gcgttcgcca aacgacaagg gaaagagatg gatagcttac gttttctcta tgatggcatt
       61 cgcatccagg cggatcaagc tccagaggac ttggatatgg aagataacga cattatcgaa
      121 gcccatcgcg aacagattgg tggcggcggt ggtgggtcgg gaggtggagg ttcagaaaat
      181 ctgtattttc agggcatgga tgtgttggct gccctggaac gtaagcacag tttaaacttg
      241 tttccgattg aaaatcgctt gtcaccccgt gctgccgccg ctctggcatc cgatgccgta
      301 aaccgttatc cgtacagtga gacggatgtg gcggtgtacg gagacgttag tgatctgaat
      361 gctgtatatg accattgcgt cagtcttacc aaggaatttt atggcgcccg tcatgcatat
      421 gttcagtttc tttccggact tcacaccatg catacagtgt taacagcagt cacaccgcca
      481 gggggccgtg taatggtcat tgcgcctgaa gacggaggac attatgcaac ggttactatt
      541 tgccaaggtt ttggctaccg cgtagagtac gtaccattcg atcgccagac tttggaaatt
      601 gactacactg ctcttgccga acgcacagcc gaacatccgg ctgatgtgat ctacttggac
      661 gcatcgacgg tattgcgcat gcctgacgcg cgcgctctgc gtgcagcagc cccaggcgct
      721 gttctgtgtc tggatgcaag tcatcttctg ggacttcttc ccgcagcccc tgggaccttg
      781 gtccttgatg ctggctttga ttcaatttct ggaagcactc acaaaacttt accgggaccc
      841 caaaagggat tgttggtgac aaactccgat gccattgccg aacaggtcgg agcgcgcatc
      901 ccttttaccg cgagttcatc gcattctgcg agcgtgggtt cgctggcgat tacattagaa
      961 gagcttttgc cccatcgcgg ggattacgca cgtcaggtga tcgcaaacgc ccgtgagctg
     1021 gctcgtcaac ttgcggcccg cggctttgac gtggcagggg aagccttcgg atttactgat
     1081 actcatcagg tgtgggtcca ccatccagag ggaaatacac cgcatgagtg gggacgtctg
     1141 ctgacagcta ctgatattcg caccactaca gtagtgcttc catcaactgc acgtagtgga
     1201 ttacgtttag gaacgcagga gttgacacgt tgggggatga aggaagacga tatgactacc
     1261 gttgcagagc ttcttgcccg tctgctttta cgcggagaac agagtcgctc agttgccgcg
     1321 gatgtacgcg acttggctcg ttcgttccca ggtgtggctt tcgcggaccg tccagcaccc
     1381 ttggcagtag cctaacttga tgggggatcc catgaggcta ggtggaggct cagtcgaaag
     1441 actgggcctt tcgttttatc tgttgtttgt cggtgaacgt ctcattttcg ccagatatcg
     1501 acgtcttaag acccactttc acatttaagt tgtttttcta atccgcatat gatcaattca
     1561 aggccgaata agaaggctgg ctctgcacct tggtgatcaa ataattcgat agcttgtcgt
     1621 aataatggcg gcatactatc agtagtaggt gtttcccttt cttctttagc gacttgatgc
     1681 tcttgatctt ccaatacgca acctaaagta aaatacccca cagcgctgag tgcatataat
     1741 gcattctcta gtgaaaaacc ttgttggcat aaaaaggcta attgtttttc gagagtttca
     1801 tactgttttt ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg atgacttagt
     1861 aaagcacatc taaaactttt agcgttatta cgtaaaaaat cttgccagct ttccccttct
     1921 aaagggcaaa agtgagtatg gtgcctatct aacatctcaa tggctaaggc gtcgagcaaa
     1981 gcccgcttat tttttacatg ccaatacaat gtaggctgct ctacacctag cttctgggcg
     2041 agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa tgcgctgtta
     2101 atcactttac ttttatctaa tctagacatc attaattcct aattgctagc attataccta
     2161 ggactgagct agccataaag ttgacactac gctctcctga gtaggacaaa tccgccgccc
     2221 tagacctagg gctttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2281 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2341 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2401 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2461 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2521 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2581 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2641 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2701 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2761 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     2821 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2881 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2941 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3001 cagtggaacg aaaactcacg ttaagggatt ttggtcatga ctagtgcttg gattctcacc
     3061 aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt
     3121 cattactgga tctatcaaca ggagtccaag cgagctctcg aaccccagag tcccgctcag
     3181 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
     3241 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
     3301 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
     3361 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
     3421 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
     3481 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
     3541 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
     3601 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
     3661 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
     3721 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
     3781 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaacctgg
     3841 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
     3901 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
     3961 tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatcttga tcccctgcgc
     4021 catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta
     4081 ccagagggcg ccccagctgg caattccgac gtctaagaaa ccattattat catgacatta
     4141 acctataaaa ataggcgtat cacgaggccc tttcgtcttc acctcgagtc cctatcagtg
     4201 atagagattg acatccctat cagtgataga gatactgagc acatcagcag gacgcactga
     4261 ccgaattcat taaagaggag aaaggtacca tgggcagcag ccatcaccat catcaccaca
     4321 tgtccctgca ggactcggag gttaaccagg aagcaaagcc ggaagtcaaa ccggaagtga
     4381 aacccgaaac tcacatcaat ctgaaggtaa gtgatggttc ttcagagata ttctttaaaa
     4441 ttaaaaaaac cacgcctctg cggcgtctta tggaa
//
