LOCUS       sequence-123078-        6096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Orthogonal GFP message under T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6096)
  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 6096)
  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: pT7oGFP
FEATURES             Location/Qualifiers
     source          1..6096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     140..159
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        140..158
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             843..890
                     /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             891..914
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(987..1004)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1157..1243
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1335..1362
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1499..2087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1988..2007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2241..2258
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      2334..2789
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2421..2440)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2631..2652
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2809..2828)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(2834..3331)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     3280..3302
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3294..3311
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3295..3311
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3327..3343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3327..3343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3340..3362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3351..3367
                     /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(3375..3381,3382..3399,3400..3405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(3454..4536)
                     /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     4498..4517
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4537..4614)
                     /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     4734..4756
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4889..4908
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5281..6096)
                     /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(5281..6096)
                     /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     6004..6023
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     join(6088..6096,1..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 actataggga gactatatct gttatttttt
      181 cacaccacag atctatgagc aaaggtgaag aactgtttac cggcgttgtg ccgattctgg
      241 tggaactgga tggcgatgtg aacggtcaca aattcagcgt gcgtggtgaa ggtgaaggcg
      301 atgccacgat tggcaaactg acgctgaaat ttatctgcac caccggcaaa ctgccggtgc
      361 cgtggccgac gctggtgacc accctgacct atggcgttca gtgttttagt cgctatccgg
      421 atcacatgaa acgtcacgat ttctttaaat ctgcaatgcc ggaaggctat gtgcaggaac
      481 gtacgattag ctttaaagat gatggcaaat ataaaacgcg cgccgttgtg aaatttgaag
      541 gcgataccct ggtgaaccgc attgaactga aaggcacgga ttttaaagaa gatggcaata
      601 tcctgggcca taaactggaa tacaacttta atagccataa tgtttatatt acggcggata
      661 aacagaaaaa tggcatcaaa gcgaatttta ccgttcgcca taacgttgaa gatggcagtg
      721 tgcagctggc agatcattat cagcagaata ccccgattgg tgatggtccg gtgctgctgc
      781 cggataatca ttatctgagc acgcagaccg ttctgtctaa agatccgaac gaaaaaggca
      841 cgcgggacca catggttctg cacgaatatg tgaatgcggc aggtattacg tggagccatc
      901 cgcagttcga aaaataagtc gaccggctgc taacaaagcc cgcggccgct gaagatcgat
      961 ctcgacgagt gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     1021 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     1081 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     1141 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1201 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     1261 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     1321 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
     1381 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccccctt
     1441 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1501 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     1561 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1621 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     1681 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1741 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1801 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1861 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1921 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1981 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2041 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2101 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2161 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     2221 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     2281 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacagatc ctgacgcgcc
     2341 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2401 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2461 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2521 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2581 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2641 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2701 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2761 ttttaacaaa atattaacgt ttacaggatc tgtatggtgc actctcagta caatctgctc
     2821 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg cctcgacctg
     2881 cagcaattcc aacgccatca aaaataattc gcgtctggcc ttcctgtagc cagctttcat
     2941 caacattaaa tgtgagcgag taacaacccg tcggattctc cgtgggaaca aacggcggat
     3001 tgaccgtaat gggataggtc acgttggtgt agatgggcgc atcgtaaccg tgcatctgcc
     3061 agtttgaggg gacgacgaca gtatcggcct caggaagatc gcactccagc cagctttccg
     3121 gcaccgcttc tggtgccgga aaccaggcaa agcgccattc gccattcagg ctgcgcaact
     3181 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     3241 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     3301 cgacggccag tgaatccgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3361 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3421 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3481 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3541 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3601 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3661 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3721 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3781 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3841 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3901 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3961 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4021 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4081 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4141 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4201 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4261 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4321 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4381 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4441 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4501 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4561 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattccatgg tgtcggaatt
     4621 gctgcaggtc gagggggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4681 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4741 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagg atggcgccca
     4801 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     4861 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     4921 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc cgtcgacctg
     4981 cagggggggg ggggcgctga ggtctgcctc gtgaagaagg tgttgctgac tcataccagg
     5041 cctgaatcgc cccatcatcc agccagaaag tgagggagcc acggttgatg agagctttgt
     5101 tgtaggtgga ccagttggtg attttgaact tttgctttgc cacggaacgg tctgcgttgt
     5161 cgggaagatg cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc
     5221 gccgtcccgt caagtcagcg taatgctctg ccagtgttac aaccaattaa ccaattctga
     5281 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     5341 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     5401 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     5461 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     5521 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     5581 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     5641 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     5701 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     5761 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     5821 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     5881 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     5941 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     6001 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     6061 cctcgagcaa gacgtttccc gttgaatatg gctcat
//
