LOCUS       sequence-68383-e        4042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BIOFAB reporter plasmid for measuring ilvGEDA termination 
            efficiency.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4042)
  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 4042)
  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: pFAB816
FEATURES             Location/Qualifiers
     source          1..4042
                     /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"
     RBS             1351..1362
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1369..1371,1372..2085)
                     /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             1369..2082
                     /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(1399..1427)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2022..2042
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2118..2189
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2205..2232
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2303..2320)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2437..2982)
                     /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(2517..2536)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3096..3190)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3221..4015)
                     /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(3332..3351)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3942..3961
                     /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 gtgattagag atcaagcctt aacgaactaa
     1201 gacccccgca ccgaaaggtc cgggggtttt ttttgacctt aaaaacataa ccgaggagca
     1261 gacaggcgga ctcactatag agggacaaac tcaaggtcat tcgcaagagt ggcctttatg
     1321 attgaccttc ttccggttaa tcaattcatt aaagaggaga aaggtaccat gagcaaagga
     1381 gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg
     1441 cacaaatttt ctgtccgtgg agagggtgaa ggtgatgcta caaacggaaa actcaccctt
     1501 aaatttattt gcactactgg aaaactacct gttccgtggc caacacttgt cactactctg
     1561 acctatggtg ttcaatgctt ttcccgttat ccggatcaca tgaaacggca tgactttttc
     1621 aagagtgcca tgcccgaagg ttatgtacag gaacgcacta tatctttcaa agatgacggg
     1681 acctacaaga cgcgtgctga agtcaagttt gaaggtgata cccttgttaa tcgtatcgag
     1741 ttaaagggta ttgattttaa agaagatgga aacattcttg gacacaaact cgagtacaac
     1801 tttaactcac acaatgtata catcacggca gacaaacaaa agaatggaat caaagctaac
     1861 ttcaaaattc gccacaacgt tgaagatggt tccgttcaac tagcagacca ttatcaacaa
     1921 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtcgacacaa
     1981 tctgtccttt cgaaagatcc caacgaaaag cgtgaccaca tggtccttct tgagtttgta
     2041 actgctgctg ggattacaca tggcatggat gagctctaca aataaggatc ggttgtcgag
     2101 taaggatctc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     2161 ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg
     2221 ggcctttctg cgtttatagg atcctaactc gagctgaagc caatagggat aacagggtaa
     2281 tcctagggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     2341 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     2401 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     2461 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     2521 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     2581 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     2641 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     2701 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     2761 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     2821 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     2881 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     2941 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
     3001 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     3061 agccccatac gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg
     3121 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
     3181 aacaggagtc caagcgagct ctcgaacccc agagtcccgc tcagaagaac tcgtcaagaa
     3241 ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc
     3301 ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct
     3361 gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt
     3421 ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg
     3481 gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt
     3541 ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat
     3601 gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg
     3661 catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc
     3721 ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag
     3781 ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt
     3841 cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca
     3901 gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata
     3961 gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa
     4021 acgatcctca tcctgtctct tg
//
