LOCUS       sequence-169790-        7948 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 7948)
  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 7948)
  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..7948
                     /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(3558..3560,3561..3563,3564..4277)
                     /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(3558..3560,3561..3563,3564..4274)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3603..3624)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3864..3883)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4211..4232
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5404..5420)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5404..5420)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5417..5439)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5428..5444
                     /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(5452..5458,5459..5476,5477..5482))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5497..5518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5635..5652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5806..6394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5886..5905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6565..7356,7357..7425))
                     /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     7188..7207
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7426..7530)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7598..7616
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7654..7676)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7776..7795
                     /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 acaaatatta ttatctactt ctatcatata ctactattca tagctagcta
     1621 cgcattgttt cctcaatcca tacgcagttg tactgttaca ttcacaaccc gtatccctca
     1681 tgacctcttg actgtatcct acagtacgta tttcctttat ttttctcttc cacgagctac
     1741 agtagccaca cccctacggt acggtacagt attgataaat caaccgacga cagcgcctct
     1801 caactgccaa acagctggaa tgcgttcgtt tgagaatgac cccaagacaa aagatccatt
     1861 ttcaacctac agtaacgtcc gcccacgttt gcaggatcca aaaatgcgag gaatcaatcc
     1921 agtcgaacta gtcgagaaag ggcaacaaag atccaatttc cgtactagac agatgtacag
     1981 tatggaatcg agtgaatcaa accagtcgat tcagtcgaac tagtcgagga ttgtaatcaa
     2041 tactgtacat agtttgcgct ttgccttcca ctaagttacc ggtacaggag tagtagattt
     2101 tgtgagattt ttcatgacaa acaaatcgaa aagtaaaaaa aaaacctatt agaaatcaac
     2161 aatgacgacg atgatgatgt aacatgatga ggaggataat cataatagtg acaattcaac
     2221 tatcgtatca ccttatttct tcacaatcag ccaaaagaaa attccctaga ggtcagcaga
     2281 aaaaattacg aaaagaacgg cgtactgtac agcaacggcg aaggattgac aaatgaatag
     2341 tattattaga ataaaaagtg tttggttttc gttatcgtta tatttatcgg tattgttact
     2401 attactattt agtttggttc tattttttaa tagtaataat aataataata atgatacaac
     2461 aatcatagta gtgggcgtta atggatttct tctaccaaca agaacaacaa taacgccgac
     2521 atcagcacag cgactactgt tgtcgtctac tacagcacaa caacaacgac tcaaagtaag
     2581 gcatagaaat aatctgcatg aaaagatcta acgatttcta tcagttcccc tacaaatgga
     2641 tgcttagggg tgcttgtatg agtgtacgaa agagccaacc aaaaaataat tttttattct
     2701 gatttttaat tttttgtgct aaaacatgct gcgctgtgta cttcctccgt gcaccggtaa
     2761 acgcgcacgg aggaaataca gtgttttaga aaaacaacgc gcaaaaatgc caatctttat
     2821 tattttgaag tgaagcgcgc tatttactgt tatcgcgcta atactgtaaa aattcgcgct
     2881 gatcttgatg ataccacctt aactttgttt ctattttttg ataacaaata caaaatctac
     2941 tagtattgtg caccctccta ctactagcta ctactaggga gtgtcgtact agtagtctaa
     3001 aaatacgcgt gttcaacatg cgcgtgctag aagtatgatg cgtgtgtaac aaatttttaa
     3061 aagattaaaa aaatctttat tttgagggat tggatgcgtg atagaatgga gaattgtgtt
     3121 cgtgggattc attttgacct accaattaaa acgaataaaa atgtcagcgt cgattataga
     3181 cacgaagtac agaccagaca gcgcgtcgac gtcagatttt tatggccaat cagattgttt
     3241 ccaacacaca aatggaaaga aagaatgcat tctgtggaag agtgtacatc cgggggaatt
     3301 tagaacgaaa accacgtcgg tgaattgtac acgcgacaat cctccaatcg tggataatcc
     3361 aggatttttt gttgatgttg aactgcatat ttgccgatcc ttcatgcgaa ctccaaattt
     3421 ctatatcatt ttttcagcaa aatccaatat ttttcagcaa aatccaaaaa acaagccatc
     3481 gttttaccgc tttcgatctt ctcataactt tattctttca acagtataaa ctaccaaaaa
     3541 caaacttata tatcaaaatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     3601 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     3661 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     3721 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     3781 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     3841 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     3901 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     3961 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     4021 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     4081 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     4141 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     4201 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     4261 acgagctgta caagtaagca ttttattaat ccttatttga tcgattcatt gattcgaata
     4321 tagtgagacc cttccgttga caactacaat cgattcctaa tttaattatt tagtagagtt
     4381 ccttgagttt ttgtcacaaa tattattatc tacttctatc atatactact attcatagct
     4441 agctacgcat tgtttcctca atccatacgc agttgtactg ttacattcac aacccgtatc
     4501 cctcatgacc tcttgactgt atcctacagt acgtatttcc tttatttttc tcttccacga
     4561 gctacagtag ccacacccct acggtacggt acagtattga taaatcaacc gacgacagcg
     4621 cctctcaact gccaaacagc tggaatgcgt tcgtttgaga atgaccccaa gacaaaagat
     4681 ccattttcaa cctacagtaa cgtccgccca cgtttgcagg atccaaaaat gcgaggaatc
     4741 aatccagtcg aactagtcga gaaagggcaa caaagatcca atttccgtac tagacagatg
     4801 tacagtatgg aatcgagtga atcaaaacag tcgattcagt cgaactagtc gaggattgta
     4861 atcaatactg tacatagttt gcgctttgcc ttccactaag ttaccggtac aggagtagta
     4921 gattttgtga gatttttcat gacaaacaaa tcgaaaagta aaaaaaaaac ctattagaaa
     4981 tcaacaatga cgacgatgat gatgtaacat gatgaggagg ataatcataa tagtgacaat
     5041 tcaactatcg tatcacctta tttcttcaca atcagccaaa agaaaattcc ctagaggtca
     5101 gcagaaaaaa ttacgaaaag aacggcgtac tgtacagcaa cggcgaagga ttgacaaatg
     5161 aatagtatta ttagaataaa aagtgtttgg ttttcgttat cgttatattt atcggtattg
     5221 ttactattac tatttagttt ggttctattt tttaatagta ataataataa taataatgat
     5281 acaacaatca tagtagtggg cgttaatgga tttcttctac caacaagaac aacaataacg
     5341 ccgacatcag cacagcgact actgttgtcg tcgactaagg catgcaagct tggcgtaatc
     5401 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5461 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5521 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5581 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5641 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5701 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5761 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5821 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5881 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5941 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6001 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6061 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6121 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6181 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6241 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6301 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6361 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6421 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6481 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6541 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6601 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6661 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6721 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6781 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6841 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6901 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6961 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7021 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7081 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7141 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7201 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7261 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7321 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7381 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7441 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7501 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7561 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7621 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     7681 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     7741 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     7801 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     7861 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     7921 gctattacgc cagctggcga aaggggga
//
