LOCUS       sequence-123074-        6155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Orthogonal GFP message under lpp5 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6155)
  AUTHORS   Orelle C, Carlson ED, Szal T, Florin T, Jewett MC, Mankin AS
  TITLE     Protein synthesis by ribosomes with tethered subunits.
  JOURNAL   Nature. 2015 Jul 29. doi: 10.1038/nature14862.
  PUBMED    26222032
REFERENCE   2  (bases 1 to 6155)
  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"; journalName: "Nature. 
            2015 Jul 29. doi: 10.1038/nature14862."
COMMENT     Sequence Label: pLpp5oGFP
FEATURES             Location/Qualifiers
     source          1..6155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    199..222
                     /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)."
     CDS             902..949
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             950..973
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(1046..1063)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1216..1302
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1394..1421
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1558..2146
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2047..2066
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2300..2317
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      2393..2848
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2480..2499)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2690..2711
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2868..2887)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(2893..3390)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     3339..3361
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3353..3370
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3354..3370
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3386..3402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3386..3402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3399..3421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3410..3426
                     /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)."
     promoter        complement(join(3434..3440,3441..3458,3459..3464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(3513..4595)
                     /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     4557..4576
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4596..4673)
                     /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."
     primer_bind     4793..4815
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4948..4967
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5340..6155)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5340..6155)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6063..6082
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     join(6147..6155,1..11)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
       61 tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt cccccccccc
      121 cctgcaggtc gacggatcct aatacgactc actatcaaaa aaatattgac aacataaaaa
      181 actttgtgtt atacttgtgg aattgtgagc ggataacaat tctatatctg ttattttttc
      241 acaccacaga tctatgagca aaggtgaaga actgtttacc ggcgttgtgc cgattctggt
      301 ggaactggat ggcgatgtga acggtcacaa attcagcgtg cgtggtgaag gtgaaggcga
      361 tgccacgatt ggcaaactga cgctgaaatt tatctgcacc accggcaaac tgccggtgcc
      421 gtggccgacg ctggtgacca ccctgaccta tggcgttcag tgttttagtc gctatccgga
      481 tcacatgaaa cgtcacgatt tctttaaatc tgcaatgccg gaaggctatg tgcaggaacg
      541 tacgattagc tttaaagatg atggcaaata taaaacgcgc gccgttgtga aatttgaagg
      601 cgataccctg gtgaaccgca ttgaactgaa aggcacggat tttaaagaag atggcaatat
      661 cctgggccat aaactggaat acaactttaa tagccataat gtttatatta cggcggataa
      721 acagaaaaat ggcatcaaag cgaattttac cgttcgccat aacgttgaag atggcagtgt
      781 gcagctggca gatcattatc agcagaatac cccgattggt gatggtccgg tgctgctgcc
      841 ggataatcat tatctgagca cgcagaccgt tctgtctaaa gatccgaacg aaaaaggcac
      901 gcgggaccac atggttctgc acgaatatgt gaatgcggca ggtattacgt ggagccatcc
      961 gcagttcgaa aaataagtcg accggctgct aacaaagccc gcggccgctg aagatcgatc
     1021 tcgacgagtg agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc
     1081 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     1141 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag
     1201 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     1261 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     1321 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     1381 catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt
     1441 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccccctta
     1501 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1561 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1621 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1681 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     1741 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1801 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1861 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1921 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     1981 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2041 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2101 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2161 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2221 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2281 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2341 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacagatcc tgacgcgccc
     2401 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2461 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2521 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2581 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2641 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2701 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2761 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2821 tttaacaaaa tattaacgtt tacaggatct gtatggtgca ctctcagtac aatctgctct
     2881 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgc ctcgacctgc
     2941 agcaattcca acgccatcaa aaataattcg cgtctggcct tcctgtagcc agctttcatc
     3001 aacattaaat gtgagcgagt aacaacccgt cggattctcc gtgggaacaa acggcggatt
     3061 gaccgtaatg ggataggtca cgttggtgta gatgggcgca tcgtaaccgt gcatctgcca
     3121 gtttgagggg acgacgacag tatcggcctc aggaagatcg cactccagcc agctttccgg
     3181 caccgcttct ggtgccggaa accaggcaaa gcgccattcg ccattcaggc tgcgcaactg
     3241 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     3301 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     3361 gacggccagt gaatccgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3421 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3481 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3541 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3601 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     3661 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     3721 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     3781 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     3841 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     3901 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     3961 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     4021 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     4081 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     4141 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     4201 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     4261 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     4321 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     4381 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     4441 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     4501 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     4561 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     4621 tccgggcgct atcatgccat accgcgaaag gttttgcacc attccatggt gtcggaattg
     4681 ctgcaggtcg agggggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4741 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4801 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagga tggcgcccaa
     4861 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     4921 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     4981 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcc gtcgacctgc
     5041 aggggggggg gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc
     5101 ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt
     5161 gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc
     5221 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg
     5281 ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac caattctgat
     5341 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     5401 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     5461 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     5521 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     5581 gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag
     5641 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     5701 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     5761 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     5821 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     5881 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     5941 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     6001 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     6061 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     6121 ctcgagcaag acgtttcccg ttgaatatgg ctcat
//
