LOCUS      pCDF-GFPplus       4442 bp DNA     circular SYN 25-AUG-2024
DEFINITION  Expression of a fast folding GFP variant GFP plus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4442)
  AUTHORS   Tong Y, Jorgensen TS, Whitford CM, Weber T, Lee SY
  TITLE     A versatile genetic engineering toolkit for E. coli based on 
            CRISPR-prime editing.
  JOURNAL   Nat Commun. 2021 Sep 1;12(1):5206. doi: 10.1038/s41467-021-25541-3.
  PUBMED    34471126
REFERENCE   2  (bases 1 to 4442)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2024 from SnapGene Server 7.0.3
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-09-1"; volume: "12(1):5206. doi"; pages: " 
            10.1038/s41467-021-25541-3"
FEATURES             Location/Qualifiers
     source          1..4442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        25..59
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (//parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     RBS             68..79
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             86..799
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(116..144)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     739..759
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      872..966
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1040..1058)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1054..1101
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1272..2063)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2064..2155)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2203..2941)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(3151..4233)
                     /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     4195..4214
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4234..4311)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4357..4376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4357..4375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4376..4400
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
ORIGIN
        1 ggttacggtt gagtaataaa tggatttacg gctagctcag tcctaggtat agtgctagct
       61 actagagaaa gaggagaaat actagatgag caaaggagaa gaacttttca ctggagttgt
      121 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga
      181 gggtgaaggt gatgctacat acggaaaact cacccttaaa tttatttgca ctactggaaa
      241 actacctgtt ccatggccaa cacttgtcac tactctgacc tatggtgttc aatgcttttc
      301 ccgttatccg gatcacatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta
      361 tgtacaggaa cgcactatat ctttcaaaga tgacgggaac tacaagacgc gtgctgaagt
      421 caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aagggtattg attttaaaga
      481 agatggaaac attctcggac acaaactaga gtacaactat aactcacaca atgtatacat
      541 cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc acaacattga
      601 agatggttcc gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc
      661 tgtcctttta ccagacaacc attacctgtc gacacaatct gccctttcga aagatcccaa
      721 cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga ttacacatgg
      781 catggatgag ctctacaaat gaagcgcata cctgcaggca tgcaagcttg cggccgcgtc
      841 gtgactggga aaaccctggc gactagtctt ggactcctgt tgatagatcc agtaatgacc
      901 tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt
      961 gagaatccag gggtccccaa taattacgat ttaaatttga cataagcctg ttcggttcga
     1021 ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1081 cgggtcttga ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg
     1141 cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc
     1201 tgccctgaac cgacgaccgg gtcatcgtgg ccggatcttg cggcccctcg gcttgaacga
     1261 attgttagac attatttgcc gactaccttg gtgatctcgc ctttcacgta gtggacaaat
     1321 tcttccaact gatctgcgcg cgaggccaag cgatcttctt cttgtccaag ataagcctgt
     1381 ctagcttcaa gtatgacggg ctgatactgg gccggcaggc gctccattgc ccagtcggca
     1441 gcgacatcct tcggcgcgat tttgccggtt actgcgctgt accaaatgcg ggacaacgta
     1501 agcactacat ttcgctcatc gccagcccag tcgggcggcg agttccatag cgttaaggtt
     1561 tcatttagcg cctcaaatag atcctgttca ggaaccggat caaagagttc ctccgccgct
     1621 ggacctacca aggcaacgct atgttctctt gcttttgtca gcaagatagc cagatcaatg
     1681 tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat tgcgctgcca ttctccaaat
     1741 tgcagttcgc gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac aacaatggtg
     1801 acttctacag cgcggagaat ctcgctctct ccaggggaag ccgaagtttc caaaaggtcg
     1861 ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac cagcaaatca
     1921 atatcactgt gtggcttcag gccgccatcc actgcggagc cgtacaaatg tacggccagc
     1981 aacgtcggtt cgagatggcg ctcgatgacg ccaactacct ctgatagttg agtcgatact
     2041 tcggcgatca ccgcttccct catactcttc ctttttcaat attattgaag catttatcag
     2101 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaatagct
     2161 agctcactcg gtcgctacgc tccgggcgtg agactgcggc gggcgctgcg gacacataca
     2221 aagttaccca cagattccgt ggataagcag gggactaaca tgtgaggcaa aacagcaggg
     2281 ccgcgccggt ggcgtttttc cataggctcc gccctcctgc cagagttcac ataaacagac
     2341 gcttttccgg tgcatctgtg ggagccgtga ggctcaacca tgaatctgac agtacgggcg
     2401 aaacccgaca ggacttaaag atccccaccg tttccggcgg gtcgctccct cttgcgctct
     2461 cctgttccga ccctgccgtt taccggatac ctgttccgcc tttctccctt acgggaagtg
     2521 tggcgctttc tcatagctca cacactggta tctcggctcg gtgtaggtcg ttcgctccaa
     2581 gctgggctgt aagcaagaac tccccgttca gcccgactgc tgcgccttat ccggtaactg
     2641 ttcacttgag tccaacccgg aaaagcacgg taaaacgcca ctggcagcag ccattggtaa
     2701 ctgggagttc gcagaggatt tgtttagcta aacacgcggt tgctcttgaa gtgtgcgcca
     2761 aagtccggct acactggaag gacagatttg gttgctgtgc tctgcgaaag ccagttacca
     2821 cggttaagca gttccccaac tgacttaacc ttcgatcaaa ccacctcccc aggtggtttt
     2881 ttcgtttaca gggcaaaaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2941 cttttctact gaaccgctct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     3001 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     3061 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3121 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3181 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3241 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3301 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3361 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3421 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3481 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     3541 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3601 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     3661 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     3721 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     3781 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     3841 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     3901 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     3961 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4021 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4081 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4141 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4201 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4261 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4321 gacgctctcc cttatgcgac tcctgcatta ggaaattaat acgactcact ataggggaat
     4381 tgtgagcgga taacaattcc cctgtagaaa taattttgtt taactttaat aaggagatat
     4441 ac
//