LOCUS       sequence-361519-        3553 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression of frSkylan-S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3553)
  AUTHORS   Osuga M, Nishimura T, Suetsugu S
  TITLE     Development of a green reversibly photoswitchable variant of Eos 
            fluorescent protein with fixation resistance.
  JOURNAL   Mol Biol Cell. 2021 Nov 1;32(21):br7. doi: 10.1091/mbc.E21-01-0044. 
            Epub 2021 Sep 8.
  PUBMED    34495704
REFERENCE   2  (bases 1 to 3553)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Cell."; date: "2021-11-1"; volume: "32(21):br7. doi"; pages: " 
            10.1091/mbc.E21-01-0044. Epub 2021 Sep 8"
COMMENT     Sequence Label: pRSET-B-frSkyran-S
FEATURES             Location/Qualifiers
     source          1..3553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             133..165
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(169..177,178..192)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             208..891
                     /codon_start=1
                     /product="fixation-resistant truly monomeric green-to-red 
                     photoswitchable fluorescent protein (Paez-Segala et al., 
                     2015)"
                     /label=mEos4b
                     /note="derived from mEos2"
                     /translation="MVSAIKPDMRIKLRMEGNVNGHHFVIDGDGTGKPYEGKQTMDLEV
                     KEGGPLPFAFDILTTAFSYGNRVFVKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNA
                     RNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALL
                     LEGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLP
                     DNARR"
     primer_bind     complement(954..972)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      968..1015
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1112..1567
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1199..1218)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1409..1430
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1593..1697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1698..1766,1767..2558)
                     /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     complement(1916..1935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2729..3317
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3218..3237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3471..3488
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
      181 gatgacgata aggatcgatg gggatccatg gtgagtgcga ttaagccaga catgaggatc
      241 aaactccgta tggaaggcaa cgtaaacggg caccactttg tgatcgacgg agatggtaca
      301 ggcaagcctt atgagggaaa acagaccatg gatcttgaag tcaaagaggg cggacctctg
      361 ccttttgcct ttgatatcct gaccactgca ttcagttacg gcaacagggt attcgtgaaa
      421 tatccagaca acatacaaga ctattttaag cagtcgtttc ctaaggggta ttcgtgggaa
      481 cgaagcttga ctttcgaaga cgggggcatt tgcaatgcca gaaacgacat aacaatggaa
      541 ggggacactt tctataataa agttcgattt tatggtacca actttcccgc caatggtcca
      601 gttatgcaga agaagacgct gaaatgggag ccctccactg agaaaatgta tgtgcgtgat
      661 ggagtgctga cgggtgatat tgagatggct ttgttgcttg aaggaaatgc ccattaccga
      721 tgtgacttca gaactactta caaagctaag gagaagggtg tcaagttacc aggcgcccac
      781 tttgtggacc acgccattga gattttaagc catgacaaag attacaacaa ggttaagctg
      841 tatgagcatg ctgttgctca ttctggattg cctgacaatg ccagacgata agaattcgaa
      901 gcttgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
      961 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1021 aggaggaact atatccggat ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     1081 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1141 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1201 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1261 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1321 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1381 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1441 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1501 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1561 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1621 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1681 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1741 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1801 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1861 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1921 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     1981 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2041 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2101 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2161 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2221 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2281 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2341 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2401 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2461 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2521 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2581 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2641 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2701 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2761 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2821 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     2881 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2941 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3001 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3061 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3121 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3181 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3241 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3301 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3361 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3421 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3481 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     3541 ttcattaatg cag
//
