LOCUS       sequence-309029-        7061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mRhubarb720 fluorescent protein and heme oxygenase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7061)
  AUTHORS   Rogers OC, Johnson DM, Firnberg E
  TITLE     mRhubarb: Engineering of monomeric, red-shifted, and brighter 
            variants of iRFP using structure-guided multi-site mutagenesis.
  JOURNAL   Sci Rep. 2019 Oct 30;9(1):15653. doi: 10.1038/s41598-019-52123-7.
  PUBMED    31666599
REFERENCE   2  (bases 1 to 7061)
  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: "Sci Rep."; 
            date: "2019-10-30"; volume: "9(1):15653. doi"; pages: " 
            10.1038/s41598-019-52123-7"
COMMENT     Sequence Label: pET-DUET-1-mRhubarb720-HO
FEATURES             Location/Qualifiers
     source          1..7061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        88..106
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    107..131
                     /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)."
     RBS             146..168
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             187..204
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             217..1167
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRHTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGQHYPASDIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLARAIGVMEE"
     promoter        1264..1282
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1283..1307
                     /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             2114..2158
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2196..2214)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2210..2257
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2294..2749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2381..2400)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2591..2612
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2864..3655,3656..3724))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3487..3506
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3725..3817)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3898..4486
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4387..4406
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4640..4657
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4672..4811
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4737..4756)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4897..4919
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4913..5104)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5676..6758)
                     /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     6720..6739
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6759..6836)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7042..7061
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
       61 tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
      121 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc
      181 agcagccatc accatcatca ccacagccag gatccgatgg ctgaaggatc cgtcgccagg
      241 cagcctgacc tcttgacctg cgacgatgag ccgatccata tccccggtgc catccaaccg
      301 catggactgc tgctcgccct cgccgccgac atgacgatcg ttgccggcag cgacaacctt
      361 cccgaactca ccggactggc gatcggcgcc ctgatcggcc gctctgcggc cgatgtcttc
      421 gactcggaga cgcacaaccg tctgacgatc gccttggccg agcccggggc ggccgtcgga
      481 gcaccgatca ctgtcggctt cacgatgcga aaggacgcag gcttcatcgg ctcctggcat
      541 cgccatgatc agctcatctt cctcgagctc gagcctcccc agcgggacgt cgccgagccg
      601 caggcgttct tccgccacac caacagcgcc atccgccgcc tgcaggccgc cgaaaccttg
      661 gagagcgcct gcgccgccgc ggcgcaagag gtgcggaaga ttaccggctt cgatcgggtg
      721 atgatctatc gcttcgcctc cgacttcagc ggcgaagtga tcgcagagga tcggtgcgcc
      781 gaggtcgagt caaaactagg ccagcactat cctgcctcag atattccggc gcaggcccgt
      841 cggctctata ccatcaaccc ggtacggatc attcccgata tcaattatcg gccggtgccg
      901 gtcaccccag acctcaatcc ggtcaccggg cggccgattg atcttagctt cgccatcctg
      961 cgcagcgtct cgcccgtcca tctggaattc atgcgcaaca taggcatgca cggcacgatg
     1021 tcgatctcga ttttgcgcgg cgagcgactg tggggattga tcgtttgcca tcaccgaacg
     1081 ccgtactacg tcgatctcga tggccgccaa gcctgcgagc tagtcgccca ggttctggcc
     1141 cgggcgatcg gcgtgatgga agagtaagag ctcggcgcgc ctgcaggtcg acaagcttgc
     1201 ggccgcataa tgcttaagtc gaacagaaag taatcgtatt gtacacggcc gcataatcga
     1261 aattaatacg actcactata ggggaattgt gagcggataa caattcccca tcttagtata
     1321 ttagttaagt ataagaagga gatatacata tgacgaatct tgcgcagaaa ctcaggtatg
     1381 gaacccaaca gagccatacc ttggcagaga acaccgcata catgaagtgc ttccttaaag
     1441 gcattgttga acgtgaacct tttagacagc tgctcgccaa tttgtactac ctttattccg
     1501 cactggaggc cgcactccgg cagcatcgcg acaatgagat catcagtgca atctattttc
     1561 ctgaactgaa tcgtacggac aagctcgcag aggatctgac gtattattac ggtccgaatt
     1621 ggcagcaaat cattcagcca acaccgtgcg ccaaaatata tgtagatcga ttgaaaacga
     1681 ttgctgcatc ggagccggaa ttgcttatcg ctcactgtta tacccgctat cttggtgatt
     1741 tgtcgggcgg ccaaagtctt aaaaacatta ttcgttcagc actgcagttg cccgaaggtg
     1801 aagggacggc tatgtatgag ttcgacagcc tgccaacacc gggagatcgc cggcagttta
     1861 aagaaatcta cagggatgtg cttaatagcc tgccactgga cgaagcaact attaatagga
     1921 tcgtagaaga agctaactat gctttttcac tgaaccgcga agtaatgcat gatctggagg
     1981 acctgatcaa agcagcgata ggcgagcata cctttgatct gcttaccaga caagatcgcc
     2041 caggttcgac cgaagctcga tcgacggcag gtcacccgat tacgcttatg tttgtgaata
     2101 actcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc
     2161 tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc
     2221 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2281 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2341 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2401 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2461 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2521 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2581 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2641 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2701 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc tggcggcacg
     2761 atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2821 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2881 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2941 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3001 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3061 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3121 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3181 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3241 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3301 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3361 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3421 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3481 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3541 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3601 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3661 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3721 tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc
     3781 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta
     3841 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3901 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3961 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4021 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4081 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4141 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4201 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4261 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4321 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4381 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4441 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4501 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4561 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4621 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4681 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     4741 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4801 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4861 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4921 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4981 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     5041 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     5101 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     5161 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     5221 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     5281 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     5341 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     5401 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     5461 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     5521 gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg ccccgcgccc
     5581 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5641 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5701 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5761 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5821 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5881 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5941 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     6001 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     6061 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     6121 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     6181 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     6241 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     6301 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     6361 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     6421 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     6481 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     6541 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     6601 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6661 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6721 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6781 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6841 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     6901 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     6961 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     7021 gcgagcccga tcttccccat cggtgatgtc ggcgatatag g
//
