LOCUS       sequence-414418-        4616 bp    DNA     circular SYN 14-APR-2025
DEFINITION  ColE1 ori, KanR, TetR, Tet promoter with a codon optimized obiH 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 4616)
  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 4616)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: P14-pZE-SUMO-ObiH
FEATURES             Location/Qualifiers
     source          1..4616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      525..571
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(618..1244)
                     /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     1149..1168
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(1257..1291)
                     /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(1488..2076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1568..1587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2164..2258)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2289..3083)
                     /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(2400..2419)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3010..3029
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3262..3280
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3301..3319,3320..3325,3326..3342,3343..3348,
                     3349..3374)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    3301..3319
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3326..3344
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             3384..3395
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3414..3431
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3762..3782
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 gcattcaaag aaatggaact gttcgggcgt gattacgcgg cccaaattgt gtcaaatgct
       61 aagaccttgg cacgtcactt gcacgagtta ggattagacg ttacggggga gagctttggg
      121 tttacccaga ctcaccaggt acacttcgct gtaggcgact tacaaaaagc cttggattta
      181 tgtgttaatt cacttcacgc agggggcatc cgtagcacga atatcgagat tcccggaaaa
      241 ccaggggtgc atggtattcg tttgggtgtg caagcgatga ctcgccgtgg catgaaggaa
      301 aaggatttcg aggtggtagc tcgtttcatt gcggatcttt acttcaagaa aactgagcca
      361 gcgaaagttg ctcagcagat taaggaattt ttgcaggcgt tcccattagc gcctctggca
      421 tattcttttg ataattattt agacgaggag ttattggctg cggtgtacca aggagcccaa
      481 cgttaacttg atgggggatc ccatggtacg cgtgctagag gcatcaaata aaacgaaagg
      541 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gtctcatttt
      601 cgccagatat cgacgtctta agacccactt tcacatttaa gttgtttttc taatccgcat
      661 atgatcaatt caaggccgaa taagaaggct ggctctgcac cttggtgatc aaataattcg
      721 atagcttgtc gtaataatgg cggcatacta tcagtagtag gtgtttccct ttcttcttta
      781 gcgacttgat gctcttgatc ttccaatacg caacctaaag taaaatgccc cacagcgctg
      841 agtgcatata atgcattctc tagtgaaaaa ccttgttggc ataaaaaggc taattgattt
      901 tcgagagttt catactgttt ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg
      961 cgatgactta gtaaagcaca tctaaaactt ttagcgttat tacgtaaaaa atcttgccag
     1021 ctttcccctt ctaaagggca aaagtgagta tggtgcctat ctaacatctc aatggctaag
     1081 gcgtcgagca aagcccgctt attttttaca tgccaataca atgtaggctg ctctacacct
     1141 agcttctggg cgagtttacg ggttgttaaa ccttcgattc cgacctcatt aagcagctct
     1201 aatgcgctgt taatcacttt acttttatct aatctagaca tcattaattc ctaattgcta
     1261 gcattatacc taggactgag ctagccataa agttgacact acgctctcct gagtaggaca
     1321 aatccgccgc cctagaccta gggctttcgg ctgcggcgag cggtatcagc tcactcaaag
     1381 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1441 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1501 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1561 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1621 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1681 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1741 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1801 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1861 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1921 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1981 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2041 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2101 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gactagtgct
     2161 tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg
     2221 gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctct cgaaccccag
     2281 agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     2341 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     2401 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     2461 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     2521 tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg
     2581 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     2641 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     2701 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     2761 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     2821 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     2881 gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca
     2941 aaaagaacct ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     3001 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     3061 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatctt
     3121 gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc
     3181 ttcccaacct taccagaggg cgccccagct ggcaattccg acgtctaaga aaccattatt
     3241 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcacctcgag
     3301 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacatcagc
     3361 aggacgcact gaccgaattc attaaagagg agaaaggtat catgggcagc agccatcacc
     3421 atcatcacca catgtccctg caggactcgg aggttaacca ggaagcaaag ccggaagtca
     3481 aaccggaagt gaaacccgaa actcacatca atctgaaggt aagtgatggt tcttcagaga
     3541 tattctttaa aattaaaaaa accacgcctc tgcggcgtct tatggaagcg ttcgccaaac
     3601 gacaagggaa agagatggat agcttacgtt ttctctatga tggcattcgc atccaggcgg
     3661 atcaagctcc agaggacttg gatatggaag ataacgacat tatcgaagcc catcgcgaac
     3721 agattggtgg cggcggtggt gggtcgggag gtggaggttc agaaaatctg tattttcagg
     3781 gctccaatgt caagcaacag acagctcaga tcgtggattg gttatcaagc actttaggta
     3841 aagaccatca gtatcgtgaa gatagcttga gtcttacagc gaacgagaac tatccgtcag
     3901 cgttggtacg tttgacgtcg ggctcgaccg caggggcgtt ttatcactgt agtttcccct
     3961 ttgaggtacc tgccggggaa tggcacttcc cggagccagg gcatatgaat gccatcgcag
     4021 accaggtacg tgatcttggg aaaacactga tcggagcaca ggcgtttgac tggcgcccaa
     4081 acggcggctc tacagcagaa caggcgttga tgttagcggc gtgcaagccc ggggaaggat
     4141 ttgtccattt cgcacaccgc gacggaggcc attttgcgct tgaatcactg gcgcagaaaa
     4201 tgggaattga aattttccac ttgccagtta accccacgag tttgcttatt gatgtggcga
     4261 aattggatga aatggtccgc cgcaatccgc acatccgtat tgtaattctg gaccagtcct
     4321 ttaagcttcg ctggcagccg ttggcggaaa ttcgttccgt actgccggat tcgtgtactt
     4381 tgacgtacga catgagtcac gatggaggtt tgattatggg tggcgttttc gattcgcctt
     4441 taagttgcgg agcagacatc gtacacggaa acacacataa gacgatccct ggtccacaga
     4501 aagggtacat cggatttaag agtgctcaac acccgctgtt agtggatacc agcctttggg
     4561 tatgccctca cctgcaatcc aactgccatg cggaacagct gccgccaatg tgggta
//
