LOCUS       sequence-183803-        7058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN Backbone for Phaeodactylum tricornutum_ fcpA promotor_NG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7058)
  AUTHORS   Serif M, Lepetit B, Weissert K, Kroth PG, Rio Bartulos C
  TITLE     A fast and reliable strategy to generate TALEN-mediated gene 
            knockouts in the diatom Phaeodactylum tricornutum
  JOURNAL   Algal Research Volume 23, April 2017, Pages 186–195
REFERENCE   2  (bases 1 to 7058)
  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: "Algal 
            Research Volume 23, April 2017, Pages 186–195"
COMMENT     Sequence Label: pM9_fcpA_NG
FEATURES             Location/Qualifiers
     source          1..7058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        516..534
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      536..545
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             545..610
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             617..637
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        join(1106..1111,1112..1129,1130..1136)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1190..1849
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1256..1275)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2191..2496
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2375..2394
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2800..3387
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     primer_bind     complement(3405..3422)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             4056..4430
                     /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"
     promoter        complement(4682..4700)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4787..4804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4958..5546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5038..5057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5717..6508,6509..6577))
                     /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     6340..6359
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6578..6682)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6750..6768
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6806..6828)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6928..6947
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7028..7046
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     7028..7045
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gggctgcagg acgcaatgga ggattatcac cgcaaaaatg aacttcgaaa aaaactttcg
       61 agcgaccatg gaaaaggagg atcagattca gattacaaca gtggattgct ctggtagcaa
      121 atatcttctg ctagattggc tcatggtcgg ttttggacgt tcgaagctca ccgtcaaaag
      181 aaacaaaaga gaagaatgac gtcttcgtga cgtagaatct acgactgtac tcggatctgg
      241 gaaatgaatt gactcacggt cttcttcgag tcctgttaca ggcccttggt ccgaaccccc
      301 acacgatttt tgcaccaaag atttgcttca atttgctgga tgttttgact gcaagatcag
      361 ctggcctagc aagagtgctc gtgttgcttc gtcgggaatc cctacgaatt tcagttctgc
      421 acaaatttgt ctgccgtttc gagaattcga tatcatcgac taattcgagc tctctggcta
      481 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta taggggccac
      541 catggactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga
      601 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
      661 cgtagatttg agaactttgg gatattcaca gcagcagcag gaaaagatca agcccaaagt
      721 gaggtcgaca gtcgcgcagc atcacgaagc gctggtgggt catgggttta cacatgccca
      781 catcgtagcc ttgtcgcagc accctgcagc ccttggcacg gtcgccgtca agtaccagga
      841 catgattgcg gcgttgccgg aagccacaca tgaggcgatc gtcggtgtgg ggaaacagtg
      901 gagcggagcc cgagcgcttg aggccctgtt gacggtcgcg ggagagctga gagggcctcc
      961 ccttcagctg gacacgggcc agttgctgaa gatcgcgaag cggggaggag tcacggcggt
     1021 cgaggcggtg cacgcgtggc gcaatgcgct cacgggagca cccctcaacc tgacagagac
     1081 cgcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg
     1141 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     1201 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     1261 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     1321 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     1381 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     1441 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     1501 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     1561 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     1621 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     1681 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     1741 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     1801 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa gatctggatc
     1861 cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag
     1921 aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt
     1981 attacagtga cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata
     2041 tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct
     2101 ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct
     2161 cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga
     2221 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
     2281 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
     2341 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
     2401 gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
     2461 aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc
     2521 cagtctgcag gtcgacggtc tcgactcacg cctgagcagg tagtggctat tgcatccaac
     2581 ggagggggca gacccgcact ggagtcaatc gtggcccagc tttcgaggcc ggaccccgcg
     2641 ctggccgcac tcactaatga tcatcttgta gcgctggcct gcctcggcgg acgacccgcc
     2701 ttggatgcgg tgaagaaggg gctcccgcac gcgcctgcat tgattaagcg gaccaacaga
     2761 aggattcccg agaggacatc acatcgagtg gcaggttccc aactcgtgaa gagtgaactt
     2821 gaggagaaaa agtcggagct gcggcacaaa ttgaaatacg taccgcatga atacatcgaa
     2881 cttatcgaaa ttgctaggaa ctcgactcaa gacagaatcc ttgagatgaa ggtaatggag
     2941 ttctttatga aggtttatgg ataccgaggg aagcatctcg gtggatcacg aaaacccgac
     3001 ggagcaatct atacggtggg gagcccgatt gattacggag tgatcgtcga cacgaaagcc
     3061 tacagcggtg ggtacaatct tcccatcggg caggcagatg agatgcaacg ttatgtcgaa
     3121 gaaaatcaga ccaggaacaa acacatcaat ccaaatgagt ggtggaaagt gtatccttca
     3181 tcagtgaccg agtttaagtt tttgtttgtc tctgggcatt tcaaaggcaa ctataaggcc
     3241 cagctcacac ggttgaatca cattacgaac tgcaatggtg cggttttgtc cgtagaggaa
     3301 ctgctcattg gtggagaaat gatcaaagcg ggaactctga cactggaaga agtcagacgc
     3361 aagtttaaca atggcgagat caatttccgc tcataaaaaa tcagcctcga ctgtgccttc
     3421 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaagcttc
     3481 agaagcgtgc tatcgaactc aaccagggac gtgcggcaca aatgggcatc cttgctctca
     3541 tggtgcacga acagttggga gtctctatcc ttccttaaaa atttaatttt cattagttgc
     3601 agtcactccg ctttggtttc acagtcagga ataacactag ctcgtcttca ccatggatgc
     3661 caatctcgcc tattcatggt gtataaaagt tcaacatcca aagctagaac ttttggaaag
     3721 agaaagaata tccgaatagg gcacggcgtg ccgtattgtt ggagtggact agcagaaagt
     3781 gaggaaggca caggatgagt tttctcgaga cataccttca gcgtcgtctt cactgtcaca
     3841 gtcaactgac agtaatcgtt gatccggaga gattcaaaat tcaatctgtt tggacctgga
     3901 taagacacaa gagcgacatc ctgacatgaa cgccgtaaac agcaaatcct ggttgaacac
     3961 gtatcctttt gggggcctcc cgctacgacg ctcgctccag ctggggcttc cttactatac
     4021 acagcgcgca tatttcacgg ttgccagatg tcaagatggc caagttgacc agtgccgttc
     4081 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
     4141 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
     4201 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
     4261 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
     4321 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
     4381 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga accttcctta
     4441 aaaatttaat tttcattagt tgcagtcact ccgctttggt ttcacagtca ggaataacac
     4501 tagctcgtct tcaccatgga tgccaatctc gcctattcat ggtgtataaa agttcaacat
     4561 ccaaagctag aacttttgga aagagaaaga atatccgaat agggcacggc gtgccgtatt
     4621 gttggagtgg actagcagaa agtgaggaag gcacaggatg agttttctcg aggccggact
     4681 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
     4741 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4801 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4861 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4921 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4981 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5041 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5101 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5161 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5221 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5281 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5341 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5401 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5461 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5521 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5581 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5641 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5701 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5761 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5821 gcttaccatc tggccccagt gctgcaatga taccgcggga cccacgctca ccggctccag
     5881 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5941 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6001 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6061 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6121 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6181 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6241 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6301 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6361 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6421 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6481 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6541 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6601 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6661 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     6721 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     6781 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     6841 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     6901 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     6961 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
     7021 acatacgatt taggtgacac tatagaacca gatccccc
//
