LOCUS       sequence-68382-e        4037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BIOFAB reporter plasmid for measuring tonB termination efficiency.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4037)
  AUTHORS   Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai 
            QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D
  TITLE     Precise and reliable gene expression via standard transcription and 
            translation initiation elements.
  JOURNAL   Nat Methods. 2013 Apr;10(4):354-60. doi: 10.1038/nmeth.2404. Epub 
            2013 Mar 10.
  PUBMED    23474465
REFERENCE   2  (bases 1 to 4037)
  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"; doi: 
            "10.1038/nmeth.2404"; journalName: "Nat Methods"; date: "2013-04"; 
            volume: "10"; issue: "4"; pages: "354-60"
COMMENT     Sequence Label: pFAB805
FEATURES             Location/Qualifiers
     source          1..4037
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(320..338,339..344,345..361,362..367,368..393)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    320..338
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    345..363
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             407..418
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             425..1102
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1198..1229
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     RBS             1346..1357
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1364..1366,1367..2080)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1364..2077
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1394..1422)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2017..2037
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2113..2184
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2200..2227
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2298..2315)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2432..2977)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2512..2531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3091..3185)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3216..4010)
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3327..3346)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3937..3956
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 atcagatcat gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac
       61 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg acgtctaggg
      121 ataacagggt aattacggcc ccagaattca aaagatctta agtaagtaag agtatacgta
      181 tatcggctaa aacgtattaa ggcgcttcgg cgcctttttt tatgggggta ttttcatccc
      241 aatccacacg tccaacgcac agcaaacacc acgtcgaccc tatcagctgc gtgctttcta
      301 tgagtcgttg ctgcatatct ccctatcagt gatagagatt gacatcccta tcagtgatag
      361 agatactgag cacatcagca ggacgcactg acctttggaa ttcattaaag aggagaaagg
      421 taccatggcg agtagcgaag acgttatcaa agagttcatg cgtttcaaag ttcgtatgga
      481 aggttccgtt aacggtcacg agttcgaaat cgaaggtgaa ggtgaaggtc gtccgtacga
      541 aggtacccag accgctaaac tgaaagttac caaaggtggt ccgctgccgt tcgcttggga
      601 catcctgtcc ccgcagttcc agtacggttc caaagcttac gttaaacacc cggctgacat
      661 cccggactac ctgaaactgt ccttcccgga aggtttcaaa tgggaacgtg ttatgaactt
      721 cgaagacggt ggtgttgtta ccgttaccca ggactcctcc ctgcaagacg gtgagttcat
      781 ctacaaagtt aaactgcgtg gtaccaactt cccgtccgac ggtccggtta tgcagaaaaa
      841 aaccatgggt tgggaagctt ccaccgaacg tatgtacccg gaagacggtg ctctgaaagg
      901 tgaaatcaaa atgcgtctga aactgaaaga cggtggtcac tacgacgctg aagttaaaac
      961 cacctacatg gctaaaaaac cggttcagct gccgggtgct tacaaaaccg acatcaaact
     1021 ggacatcacc tcccacaacg aagactacac catcgttgaa cagtacgaac gtgctgaagg
     1081 tcgtcactcc accggtgctt aataatctgc tgagtgatag actcaaggtc gctcctagcg
     1141 agtggccttt atgattatca ctttacttat gtgatcaccg aaattcagta agcagaaagt
     1201 caaaagcctc cgaccggagg cttttgacta ttactcaaca ggtaaggcgc gaggttttcg
     1261 gcggactcac tatagaggga caaactcaag gtcattcgca agagtggcct ttatgattga
     1321 ccttcttccg gttaatcaat tcattaaaga ggagaaaggt accatgagca aaggagaaga
     1381 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
     1441 attttctgtc cgtggagagg gtgaaggtga tgctacaaac ggaaaactca cccttaaatt
     1501 tatttgcact actggaaaac tacctgttcc gtggccaaca cttgtcacta ctctgaccta
     1561 tggtgttcaa tgcttttccc gttatccgga tcacatgaaa cggcatgact ttttcaagag
     1621 tgccatgccc gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaccta
     1681 caagacgcgt gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa
     1741 gggtattgat tttaaagaag atggaaacat tcttggacac aaactcgagt acaactttaa
     1801 ctcacacaat gtatacatca cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa
     1861 aattcgccac aacgttgaag atggttccgt tcaactagca gaccattatc aacaaaatac
     1921 tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcga cacaatctgt
     1981 cctttcgaaa gatcccaacg aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc
     2041 tgctgggatt acacatggca tggatgagct ctacaaataa ggatcggttg tcgagtaagg
     2101 atctccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     2161 tgttgtttgt cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct
     2221 ttctgcgttt ataggatcct aactcgagct gaagccaata gggataacag ggtaatccta
     2281 gggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga
     2341 gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac
     2401 agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc
     2461 atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc
     2521 aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg
     2581 gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg
     2641 taggcagttc gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc
     2701 gccttatccg gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg
     2761 gcagcagcca ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg
     2821 ctaaactgaa aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa
     2881 agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc
     2941 agagcaagag attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat
     3001 aaaatatttc tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc
     3061 catacgatat aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca
     3121 accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag
     3181 gagtccaagc gagctctcga accccagagt cccgctcaga agaactcgtc aagaaggcga
     3241 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     3301 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     3361 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     3421 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg
     3481 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     3541 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     3601 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     3661 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     3721 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     3781 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc
     3841 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     3901 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     3961 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     4021 cctcatcctg tctcttg
//
