LOCUS       sequence-213981-        5333 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Translation reporter with inducible translation coupling (low native
            GFP expression, high under coupling).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5333)
  AUTHORS   Cambray G, Guimaraes JC, Arkin AP
  TITLE     Evaluation of 244,000 synthetic sequences reveals design principles 
            to optimize translation in Escherichia coli.
  JOURNAL   Nat Biotechnol. 2018 Nov;36(10):1005-1015. doi: 10.1038/nbt.4238. 
            Epub 2018 Sep 24.
  PUBMED    30247489
REFERENCE   2  (bases 1 to 5333)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt.4238"; 
            journalName: "Nat Biotechnol"; date: "2018-11"; volume: "36"; issue:
            "10"; pages: "1005-1015"
COMMENT     Sequence Label: pGC4785
FEATURES             Location/Qualifiers
     source          1..5333
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(4..75)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(165..842)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             complement(877..1959)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1921..1940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1960..2037)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     RBS             2326..2334
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             2473..2490
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2500..2520
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2557..3267
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLAEHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     3207..3227
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3303..3374
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3390..3417
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(3581..4169)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3661..3680)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4257..4351)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4382..5176)
                     /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(4493..4512)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5103..5122
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt ccgactgagc
       61 ctttcgtttt atttgatgcc tggcagttcc catcacataa gtaaagtgat aatcataaag
      121 gccactcgct aggagcgacc ttgagtctat cactcagcag attattaagc accggtggag
      181 tgacgacctt cagcacgttc gtactgttca acgatggtgt agtcttcgtt gtgggaggtg
      241 atgtccagtt tgatgtcggt tttgtaagca cccggcagct gaaccggttt tttagccatg
      301 taggtggttt taacttcagc gtcgtagtga ccaccgtctt tcagtttcag acgcattttg
      361 atttcacctt tcagagcacc gtcttccggg tacatacgtt cggtggaagc ttcccaaccc
      421 atggtttttt tctgcataac cggaccgtcg gacgggaagt tggtgccacg cagtttaact
      481 ttgtagatga actcaccgtc ttgcagggag gagtcctggg taacggtaac aacaccaccg
      541 tcttcgaagt tcataacacg ttcccatttg aaaccttccg ggaaggacag tttcaggtag
      601 tccgggatgt cagccgggtg tttaacgtaa gctttggaac cgtactggaa ctgcggggac
      661 aggatgtccc aagcgaacgg cagcggacca cctttggtaa ctttcagttt agcggtctgg
      721 gtgccttcgt acggacgacc ttcaccttca ccttcgattt cgaactcgtg accgttaacg
      781 gaaccttcca tacgaacttt gaaacgcatg aactctttga taacgtcttc gctactcgcc
      841 ataccacaac tcccaaaaaa gatattttaa tctagattac tgcccgcttt ccagtcggga
      901 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
      961 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     1021 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     1081 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     1141 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     1201 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     1261 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     1321 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     1381 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     1441 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     1501 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     1561 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     1621 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     1681 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     1741 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     1801 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     1861 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     1921 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     1981 ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcaga
     2041 tcttaagtaa gtaagagtat acgtatatcg gctaataacg tattaaggcg cttcggcgcc
     2101 tttttttatg ggggtatttt catcccaatc cacacgtcca acgcacagca aacaccacgt
     2161 cgaccctatc agctgcgtgc tttctatgag tcgttgctgc ataacaaaaa gagtattgac
     2221 ttattgtgag cggataacat ataatgtgtg gagggcccaa gttcacttaa aaaggagatc
     2281 aacaatgaaa gcaatttagg tactgaaaca tcttaatcat gcacataagg aggtaccata
     2341 atgatcggcc ctctcttatg tctgcatcac gtgaaagctg ttcacgttgc aacactagtt
     2401 cagagtctga attacccgaa atcttctccg gaatctcgtg gtggatccgg tggatcgggc
     2461 gggtcatact accatcacca ccaccaccat ttggagtcgg aaaatttata cttccaatcc
     2521 ggttccgccg gatctgctgc cggttccggt gaatttagca aaggagaaga acttttcacc
     2581 ggagtagtcc cgattctggt tgaattagat ggtgatgtta atgggcacaa attttctgtc
     2641 cgtggagagg gtgaaggtga tgctacaaac ggaaaactca cccttaaatt catttgcaca
     2701 acgggaaaac taccagtacc gtggccaaca ctggtcacta ctctgaccta tggtgttcaa
     2761 tgcttttccc gttatccgga tcacatgaaa cggcatgact ttttcaagag tgccatgccc
     2821 gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaccta caagacgcgt
     2881 gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa gggtattgat
     2941 tttaaagaag atggaaacat tcttggacac aaactcgagt acaactttaa ctcacacaat
     3001 gtatacatca cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattcgccac
     3061 aacgttgaag atggttccgt tcaactagct gagcattatc aacaaaatac tccaattgga
     3121 gatggacctg tccttttacc agacaaccat tacctgtcga cacaatctgt cctttcgaaa
     3181 gatcccaacg aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc tgctgggatt
     3241 acacatggca tggatgagct ctacaaataa ggatcggttg tcgagtaagg atctccaggc
     3301 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     3361 cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt
     3421 ataaagcttc ctagggcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3481 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3541 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3601 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3661 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3721 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3781 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3841 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3901 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3961 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4021 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4081 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4141 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4201 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgactagt gcttggattc
     4261 tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct
     4321 gaggtcatta ctggatctat caacaggagt ccaagcgagc tctcgaaccc cagagtcccg
     4381 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     4441 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     4501 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     4561 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     4621 cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg
     4681 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     4741 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     4801 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     4861 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     4921 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     4981 gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     5041 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     5101 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     5161 catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat catgatcccc
     5221 tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag ggcttcccaa
     5281 ccttaccaga gggcgcccca gctggcaatt ccgaattcta tagtgagtcc gcc
//
