LOCUS       sequence-167038-        7946 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ShBle and EGFP with FCP promoters/terminators for F. 
            cylindrus transformation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7946)
  AUTHORS   Faktorova D, Nisbet RER, Fernandez Robledo JA, Casacuberta E, Sudek 
            L, Allen AE, Ares M Jr, Areste C, Balestreri C, Barbrook AC, 
            Beardslee P, Bender S, Booth DS, Bouget FY, Bowler C, Breglia SA, 
            Brownlee C, Burger G, Cerutti H, Cesaroni R, Chiurillo MA, Clemente 
            T, Coles DB, Collier JL, Cooney EC, Coyne K, Docampo R, Dupont CL, 
            Edgcomb V, Einarsson E, Elustondo PA, Federici F, Freire-Beneitez V,
            Freyria NJ, Fukuda K, Garcia PA, Girguis PR, Gomaa F, Gornik SG, Guo
            J, Hampl V, Hanawa Y, Haro-Contreras ER, Hehenberger E, Highfield A,
            Hirakawa Y, Hopes A, Howe CJ, Hu I, Ibanez J, Irwin NAT, Ishii Y, 
            Janowicz NE, Jones AC, Kachale A, Fujimura-Kamada K, Kaur B, Kaye 
            JZ, Kazana E, Keeling PJ, King N, Klobutcher LA, Lander N, Lassadi 
            I, Li Z, Lin S, Lozano JC, Luan F, Maruyama S, Matute T, Miceli C, 
            Minagawa J, Moosburner M, Najle SR, Nanjappa D, Nimmo IC, Noble L, 
            Novak Vanclova AMG, Nowacki M, Nunez I, Pain A, Piersanti A, 
            Pucciarelli S, Pyrih J, Rest JS, Rius M, Robertson D, Ruaud A, 
            Ruiz-Trillo I, Sigg MA, Silver PA, Slamovits CH, Jason Smith G, 
            Sprecher BN, Stern R, Swart EC, Tsaousis AD, Tsypin L, Turkewitz A, 
            Turnsek J, Valach M, Verge V, von Dassow P, von der Haar T, Waller 
            RF, Wang L, Wen X, Wheeler G, Woods A, Zhang H, Mock T, Worden AZ, 
            Lukes J
  TITLE     Genetic tool development in marine protists: emerging model 
            organisms for experimental cell biology.
  JOURNAL   Nat Methods. 2020 May;17(5):481-494. doi: 10.1038/s41592-020-0796-x.
            Epub 2020 Apr 6.
  PUBMED    32251396
REFERENCE   2  (bases 1 to 7946)
  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"; doi: 
            "10.1038/s41592-020-0796-x"; journalName: "Nat Methods"; date: 
            "2020-05"; volume: "17"; issue: "5"; pages: "481-494"
COMMENT     Sequence Label: pUC:FCP:ShBle:FCP:EGFP
FEATURES             Location/Qualifiers
     source          1..7946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..63
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     55..72
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     56..72
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1078..1452
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     CDS             join(3557..3559,3560..3562,3563..4276)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3557..3559,3560..3562,3563..4273)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3602..3623)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3863..3882)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4210..4231
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5402..5418)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5402..5418)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5415..5437)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5426..5442
                     /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(5450..5456,5457..5474,5475..5480))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5495..5516
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5633..5650)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5804..6392)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5884..5903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6563..7354,7355..7423))
                     /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     7186..7205
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7424..7528)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7596..7614
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7652..7674)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7774..7793
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
       61 acgacggcca gtgaattcga gctcggtacc ccaaagtaag gcatagaaat aatctgcatg
      121 aaaagatcta acgatttcta tcagttcccc tacaaatgga tgcttagggg tgcttgtatg
      181 agtgtacgaa agagccaacc aaaaaataat tttttattct gatttttaat tttttgtgct
      241 aaaacatgct gcgctgtgta cttcctccgt gcaccggtaa acgcgcacgg aggaaataca
      301 gtgttttaga aaaacaacgc gcaaaaatgc caatctttat tattttgaag tgaagcgcgc
      361 tatttactgt tatcgcgcta atactgtaaa aattcgcgct gatcttgatg ataccacctt
      421 aactttgttt ctattttttg ataacaaata caaaatctac tagtattgtg caccctccta
      481 ctactagcta ctactaggga gtgtcgtact agtagtctaa aaatacgcgt gttcaacatg
      541 cgcgtgctag aagtatgatg cgtgtgtaac aaatttttaa aagattaaaa aaatctttat
      601 tttgagggat tggatgcgtg atagaatgga gaattgtgtt cgtgggattc attttgacct
      661 accaattaaa acgaataaaa atgtcagcgt cgattataga cacgaagtac agaccagaca
      721 gcgcgtcgac gtcagatttt tatggccaat cagattgttt ccaacacaca aatggaaaga
      781 aagaatgcat tctgtggaag agtgtacatc cgggggaatt tagaacgaaa accacgtcgg
      841 tgaattgtac acgcgacaat cctccaatcg tggataatcc aggatttttt gttgatgttg
      901 aactgcatat ttgccgatcc ttcatgcgaa ctccaaattt ctatatcatt ttttcagcaa
      961 aatccaatat ttttcagcaa aatccaaaaa acaagccatc gttttaccgc tttcgatctt
     1021 ctcataactt tattctttca acagtataaa ctaccaaaaa caaacttata tatcaaaatg
     1081 gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag
     1141 ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg
     1201 gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac
     1261 accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc
     1321 gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg
     1381 tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag
     1441 gagcaggact gagcatttta ttaatcctta tttgatcgat tcattgattc gaatatagtg
     1501 agacccttcc gttgacaact acaatcgatt cctaatttaa ttatttagta gagttccttg
     1561 agtttttgtc acaaatatat tatctacttc tatcatatac tactattcat agctagctac
     1621 gcattgtttc ctcaatccat acgcagttgt actgttacat tcacaacccg tatccctcat
     1681 gacctcttga ctgtatccta cagtacgtat ttcctttatt tttctcttcc acgagctaca
     1741 gtagccacac ccctacggta cggtacagta ttgataaatc aaccgacgac agcgcctctc
     1801 aactgccaaa cagctggaat gcgttcgttt gagaatgacc ccaagacaaa agatccattt
     1861 tcaacctaca gtaacgtccg cccacgtttg caggatccaa aaatgcgagg aatcaatcca
     1921 gtcgaactag tcgagaaagg gcaacaaaga tccaatttcc gtactagaca gatgtacagt
     1981 atggaatcga gtgaatcaaa ccagtcgatt cagtcgaact agtcgaggat tgtaatcaat
     2041 actgtacata gtttgcgctt tgccttccac taagttaccg gtacaggagt agtagatttt
     2101 gtgagatttt tcatgacaaa caaatcgaaa agtaaaaaaa aaacctatta gaaatcaaca
     2161 atgacgacga tgatgatgta acatgatgag gaggataatc ataatagtga caattcaact
     2221 atcgtatcac cttatttctt cacaatcagc caaaagaaaa ttccctagag gtcagcagaa
     2281 aaaattacga aaagaacggc gtactgtaca gcaacggcga aggattgaca aatgaatagt
     2341 attattagaa taaaaagtgt ttggttttcg ttatcgttat atttatcggt attgttacta
     2401 ttactattta gtttggttct attttttaat agtaataata ataataataa tgatacaaca
     2461 atcatagtag tgggcgttaa tggatttctt ctaccaacaa gaacaacaat aacgccgaca
     2521 tcagcacagc gactactgtt gtcgtctact acagcacaac aacaacgact caaagtaagg
     2581 catagaaata atctgcatga aaagatctaa cgatttctat cagttcccct acaaatggat
     2641 gcttaggggt gcttgtatga gtgtacgaaa gagccaacca aaaaataatt ttttattctg
     2701 atttttaatt ttttgtgcta aaacatgctg cgctgtgtac ttcctccgtg caccggtaaa
     2761 cgcgcacgga ggaaatacag tgttttagaa aaacaacgcg caaaaatgcc aatctttatt
     2821 attttgaagt gaagcgcgct atttactgtt atcgcgctaa tactgtaaaa attcgcgctg
     2881 atcttgatga taccacctta actttgtttc tattttttga taacaaatac aaaatctact
     2941 agtattgtgc accctcctac tactagctac tactagggag tgtcgtacta gtagtctaaa
     3001 aatacgcgtg ttcaacatgc gcgtgctaga agtatgatgc gtgtgtaaca aatttttaaa
     3061 agattaaaaa aatctttatt ttgagggatt ggatgcgtga tagaatggag aattgtgttc
     3121 gtgggattca ttttgaccta ccaattaaaa cgaataaaaa tgtcagcgtc gattatagac
     3181 acgaagtaca gaccagacag cgcgtcgacg tcagattttt atggccaatc agattgtttc
     3241 caacacacaa atggaaagaa agaatgcatt ctgtggaaga gtgtacatcc gggggaattt
     3301 agaacgaaaa ccacgtcggt gaattgtaca cgcgacaatc ctccaatcgt ggataatcca
     3361 ggattttttg ttgatgttga actgcatatt tgccgatcct tcatgcgaac tccaaatttc
     3421 tatatcattt tttcagcaaa atccaatatt tttcagcaaa atccaaaaaa caagccatcg
     3481 ttttaccgct ttcgatcttc tcataacttt attctttcaa cagtataaac taccaaaaac
     3541 aaacttatat atcaaaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     3601 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     3661 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     3721 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     3781 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3841 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     3901 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3961 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     4021 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     4081 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     4141 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     4201 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     4261 cgagctgtac aagtaagcat tttattaatc cttatttgat cgattcattg attcgaatat
     4321 agtgagaccc ttccgttgac aactacaatc gattcctaat ttaattattt agtagagttc
     4381 cttgagtttt tgtcacaaat atattatcta cttctatcat atactactat tcatagctag
     4441 ctacgcattg tttcctcaat ccatacgcag ttgtactgtt acattcacaa cccgtatccc
     4501 tcatgacctc ttgactgtat cctacagtac gtatttcctt tatttttctc ttccacgagc
     4561 tacagtagcc acacccctac ggtacggtac agtattgata aatcaaccga cgacagcgcc
     4621 tctcaactgc caaacagctg gaatgcgttc gtttgagaat gaccccaaga caaaagatcc
     4681 attttcaacc tacagtaacg tccgcccacg tttgcaggat ccaaaaatgc gaggaatcaa
     4741 tccagtcgaa ctagtcgaga aagggcaaca aagatccaat ttccgtacta gacagatgta
     4801 cagtatggaa tcgagtgaat caaaccagtc gattcagtcg aactagtcga ggattgtaat
     4861 caatactgta catagtttgc gctttgcctt ccactaagtt accggtacag gagtagtaga
     4921 ttttgtgaga tttttcatga caaacaaatc gaaaagtaaa aaaaaaacct attagaaatc
     4981 aacaatgacg acgatgatga tgtaacatga tgaggaggat aatcataata gtgacaattc
     5041 aactatcgta tcaccttatt tcttcacaat cagccaaaag aaaattccct agaggtcagc
     5101 agaaaaaatt acgaaaagaa cggcgtactg tacagcaacg gcgaaggatt gacaaatgaa
     5161 tagtattatt agaataaaaa gtgtttggtt ttcgttatcg ttatatttat cggtattgtt
     5221 actattacta tttagtttgg ttctattttt taatagtaat aataataata ataatgatac
     5281 aacaatcata gtagtgggcg ttaatggatt tcttctacca acaagaacaa caataacgcc
     5341 gacatcagca cagcgactac tgttgtcgtc tactaaggca tgcaagcttg gcgtaatcat
     5401 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5461 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5521 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5581 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5641 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5701 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5761 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5821 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5881 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5941 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6001 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6061 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6121 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6181 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6241 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6301 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6361 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6421 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6481 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6541 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6601 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6661 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6721 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6781 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6841 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6901 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6961 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7021 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7081 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7141 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7201 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7261 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7321 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7381 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7441 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7501 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     7561 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     7621 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     7681 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     7741 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     7801 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     7861 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     7921 tattacgcca gctggcgaaa ggggga
//
