LOCUS       sequence-175097-        7057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN Backbone for Phaeodactylum tricornutum_ fcpA promotor_NI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7057)
  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 7057)
  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_NI
FEATURES             Location/Qualifiers
     source          1..7057
                     /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             4055..4429
                     /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(4681..4699)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4786..4803)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4957..5545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5037..5056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5716..6507,6508..6576))
                     /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     6339..6358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6577..6681)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6749..6767
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6805..6827)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6927..6946
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7027..7045
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     7027..7044
                     /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 atcgggggca 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 gctacgacgc tcgctccagc tggggcttcc ttactataca
     4021 cagcgcgcat atttcacggt tgccagatgt caagatggcc aagttgacca gtgccgttcc
     4081 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt
     4141 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt
     4201 catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg
     4261 cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc
     4321 ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg
     4381 cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgaa ccttccttaa
     4441 aaatttaatt ttcattagtt gcagtcactc cgctttggtt tcacagtcag gaataacact
     4501 agctcgtctt caccatggat gccaatctcg cctattcatg gtgtataaaa gttcaacatc
     4561 caaagctaga acttttggaa agagaaagaa tatccgaata gggcacggcg tgccgtattg
     4621 ttggagtgga ctagcagaaa gtgaggaagg cacaggatga gttttctcga ggccggactc
     4681 cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca
     4741 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4801 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4861 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4921 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4981 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5041 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5101 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     5161 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5221 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5281 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5341 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5401 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5461 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5521 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5581 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5641 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5701 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5761 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5821 cttaccatct ggccccagtg ctgcaatgat accgcgggac ccacgctcac cggctccaga
     5881 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5941 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6001 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6061 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6121 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6181 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6241 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6301 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6361 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6421 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6481 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6541 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6601 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6661 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     6721 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     6781 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     6841 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     6901 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     6961 tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca
     7021 catacgattt aggtgacact atagaaccag atccccc
//
