LOCUS       sequence-68386-e        4035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BIOFAB reporter plasmid for measuring rnpB T1 termination 
            efficiency.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4035)
  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 4035)
  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: pFAB815
FEATURES             Location/Qualifiers
     source          1..4035
                     /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             1344..1355
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1362..1364,1365..2078)
                     /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             1362..2075
                     /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(1392..1420)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2015..2035
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2111..2182
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2198..2225
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2296..2313)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2430..2975)
                     /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(2510..2529)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3089..3183)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3214..4008)
                     /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(3325..3344)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3935..3954
                     /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 gtgattcggt cagtttcacc tgatttacgt
     1201 aaaaacccgc ttcggcgggt ttttgctttt ggaggggcag aaagatgaat gactgtcggc
     1261 ggactcacta tagagggaca aactcaaggt cattcgcaag agtggccttt atgattgacc
     1321 ttcttccggt taatcaattc attaaagagg agaaaggtac catgagcaaa ggagaagaac
     1381 ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
     1441 tttctgtccg tggagagggt gaaggtgatg ctacaaacgg aaaactcacc cttaaattta
     1501 tttgcactac tggaaaacta cctgttccgt ggccaacact tgtcactact ctgacctatg
     1561 gtgttcaatg cttttcccgt tatccggatc acatgaaacg gcatgacttt ttcaagagtg
     1621 ccatgcccga aggttatgta caggaacgca ctatatcttt caaagatgac gggacctaca
     1681 agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt taatcgtatc gagttaaagg
     1741 gtattgattt taaagaagat ggaaacattc ttggacacaa actcgagtac aactttaact
     1801 cacacaatgt atacatcacg gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa
     1861 ttcgccacaa cgttgaagat ggttccgttc aactagcaga ccattatcaa caaaatactc
     1921 caattggcga tggccctgtc cttttaccag acaaccatta cctgtcgaca caatctgtcc
     1981 tttcgaaaga tcccaacgaa aagcgtgacc acatggtcct tcttgagttt gtaactgctg
     2041 ctgggattac acatggcatg gatgagctct acaaataagg atcggttgtc gagtaaggat
     2101 ctccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     2161 ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt
     2221 ctgcgtttat aggatcctaa ctcgagctga agccaatagg gataacaggg taatcctagg
     2281 gatatattcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc
     2341 ggaaatggct tacgaacggg gcggagattt cctggaagat gccaggaaga tacttaacag
     2401 ggaagtgaga gggccgcggc aaagccgttt ttccataggc tccgcccccc tgacaagcat
     2461 cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag
     2521 gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt
     2581 gtcattccgc tgttatggcc gcgtttgtct cattccacgc ctgacactca gttccgggta
     2641 ggcagttcgc tccaagctgg actgtatgca cgaacccccc gttcagtccg accgctgcgc
     2701 cttatccggt aactatcgtc ttgagtccaa cccggaaaga catgcaaaag caccactggc
     2761 agcagccact ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct
     2821 aaactgaaag gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag
     2881 agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag
     2941 agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aatcagataa
     3001 aatatttcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca
     3061 tacgatataa gttgttacta gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac
     3121 cgagcgttct gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga
     3181 gtccaagcga gctctcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata
     3241 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     3301 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     3361 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     3421 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg
     3481 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     3541 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     3601 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     3661 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     3721 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     3781 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag
     3841 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     3901 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     3961 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
     4021 tcatcctgtc tcttg
//
