LOCUS       sequence-183832-        7012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN Backbone for Phaeodactylum tricornutum_ NR promotor_NI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7012)
  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 7012)
  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_NR_NI
FEATURES             Location/Qualifiers
     source          1..7012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        491..509
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      511..520
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             520..585
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             592..612
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        join(1081..1086,1087..1104,1105..1111)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1165..1824
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1231..1250)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2166..2471
                     /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     2350..2369
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2775..3362
                     /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(3380..3397)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             3907..4476
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4730..4748)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4835..4852)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5006..5594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5086..5105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5765..6556,6557..6625))
                     /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     6388..6407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6626..6730)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6798..6816
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6854..6876)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6976..6995
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcggaagtga ctgtaaacga gaagtgcacg aagccttttc ttgtgacgtc acaaaccgaa
       61 cagccctacg tggcgtgcga ctttccggca ctttggatat cgttgcccta tatgtattgg
      121 gtgtatacat gtcgtattcc aactacgaca gaaaacagtc agttacgggt attcgaactt
      181 ccggcacctg cagcgaggag ttttgtgtcc gtcgcacatc ctccgtgctt tcggcaccta
      241 cgtaggcggc aaacttcctg ccttccgccc gccttgcggc attccggctc cgggccagaa
      301 ttgcccgggt gttcacaatt ttgcctcctc acgaaaaaac gttctacttt gtattttggt
      361 cgggtttcgg atccttccag caactattct cattcaaagt caccacttgt gcgaacggaa
      421 ttcgatatca tcgactaatt cgagctctct ggctaactag agaacccact gcttactggc
      481 ttatcgaaat taatacgact cactataggg gccaccatgg actataagga ccacgacgga
      541 gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag
      601 aagcggaagg tcggtatcca cggagtccca gcagccgtag atttgagaac tttgggatat
      661 tcacagcagc agcaggaaaa gatcaagccc aaagtgaggt cgacagtcgc gcagcatcac
      721 gaagcgctgg tgggtcatgg gtttacacat gcccacatcg tagccttgtc gcagcaccct
      781 gcagcccttg gcacggtcgc cgtcaagtac caggacatga ttgcggcgtt gccggaagcc
      841 acacatgagg cgatcgtcgg tgtggggaaa cagtggagcg gagcccgagc gcttgaggcc
      901 ctgttgacgg tcgcgggaga gctgagaggg cctccccttc agctggacac gggccagttg
      961 ctgaagatcg cgaagcgggg aggagtcacg gcggtcgagg cggtgcacgc gtggcgcaat
     1021 gcgctcacgg gagcacccct caacctgaca gagaccgcgg ccgcattagg caccccaggc
     1081 tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga tccgtcgaga
     1141 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
     1201 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
     1261 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
     1321 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
     1381 ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
     1441 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     1501 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
     1561 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     1621 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
     1681 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     1741 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     1801 gatgagtggc agggcggggc gtaaagatct ggatccggct tactaaaagc cagataacag
     1861 tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga
     1921 agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca
     1981 gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat
     2041 gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat
     2101 ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg
     2161 gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg
     2221 atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg
     2281 cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg
     2341 aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag
     2401 aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct
     2461 ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga cggtctcgac
     2521 tcacgcctga gcaggtagtg gctattgcat ccaacatcgg gggcagaccc gcactggagt
     2581 caatcgtggc ccagctttcg aggccggacc ccgcgctggc cgcactcact aatgatcatc
     2641 ttgtagcgct ggcctgcctc ggcggacgac ccgccttgga tgcggtgaag aaggggctcc
     2701 cgcacgcgcc tgcattgatt aagcggacca acagaaggat tcccgagagg acatcacatc
     2761 gagtggcagg ttcccaactc gtgaagagtg aacttgagga gaaaaagtcg gagctgcggc
     2821 acaaattgaa atacgtaccg catgaataca tcgaacttat cgaaattgct aggaactcga
     2881 ctcaagacag aatccttgag atgaaggtaa tggagttctt tatgaaggtt tatggatacc
     2941 gagggaagca tctcggtgga tcacgaaaac ccgacggagc aatctatacg gtggggagcc
     3001 cgattgatta cggagtgatc gtcgacacga aagcctacag cggtgggtac aatcttccca
     3061 tcgggcaggc agatgagatg caacgttatg tcgaagaaaa tcagaccagg aacaaacaca
     3121 tcaatccaaa tgagtggtgg aaagtgtatc cttcatcagt gaccgagttt aagtttttgt
     3181 ttgtctctgg gcatttcaaa ggcaactata aggcccagct cacacggttg aatcacatta
     3241 cgaactgcaa tggtgcggtt ttgtccgtag aggaactgct cattggtgga gaaatgatca
     3301 aagcgggaac tctgacactg gaagaagtca gacgcaagtt taacaatggc gagatcaatt
     3361 tccgctcata aaaaatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3421 ccctcccccg tgccttcctt gaccctggaa gcttcaattt actttaagac tagcgcaatt
     3481 tttttcttac attagatgaa cataaaacga cgatgaggat cactgttagg gaaacgatga
     3541 actacgctga ggacttccta ttattcgtcc tttgatggcg tggttgttct ttggtttgcg
     3601 gccgagcgca acgaccaagc cctgtaccaa aaacgaagtt gataatcaga tatttgagtt
     3661 gtaacaagaa actaaagcat tattttagca gtagcacgaa tcaggatcta aaatgaacgc
     3721 atccggagag attcaaaatt caatctgttt ggacctggat aagacacaag agcgacatcc
     3781 tgacatgaac gccgtaaaca gcaaatcctg gttgaacacg tatccttttg ggggcctccc
     3841 gctacgacgc tcgctccagc tggggcttcc ttactataca cagcgcgcat atttcacggt
     3901 tgccagatga ccactcttga cgacacggct taccggtacc gcaccagtgt cccgggggac
     3961 gccgaggcca tcgaggcact ggatgggtcc ttcaccaccg acaccgtctt ccgcgtcacc
     4021 gccaccgggg acggcttcac cctgcgggag gtgccggtgg acccgcccct gaccaaggtg
     4081 ttccccgacg acgaatcgga cgacgaatcg gacgacgggg aggacggcga cccggactcc
     4141 cggacgttcg tcgcgtacgg ggacgacggc gacctggcgg gcttcgtggt cgtctcgtac
     4201 tccggctgga accgccggct gaccgtcgag gacatcgagg tcgccccgga gcaccggggg
     4261 cacggggtcg ggcgcgcgtt gatggggctc gcgacggagt tcgcccgcga gcggggcgcc
     4321 gggcacctct ggctggaggt caccaacgtc aacgcaccgg cgatccacgc gtaccggcgg
     4381 atggggttca ccctctgcgg cctggacacc gccctgtacg acggcaccgc ctcggacggc
     4441 gagcaggcgc tctacatgag catgccctgc ccctgaaaac cttccttaaa aatttaattt
     4501 tcattagttg cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc
     4561 accatggatg ccaatctcgc ctattcatgg tgtataaaag ttcaacatcc aaagctagaa
     4621 cttttggaaa gagaaagaat atccgaatag ggcacggcgt gccgtattgt tggagtggac
     4681 tagcagaaag tgaggaaggc acaggatgag ttttctcgag gccggactcc ctatagtgag
     4741 tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga
     4801 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4861 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4921 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4981 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5041 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5101 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5161 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5221 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5281 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5341 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5401 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5461 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5521 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5581 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5641 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5701 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5761 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5821 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5881 gccccagtgc tgcaatgata ccgcgggacc cacgctcacc ggctccagat ttatcagcaa
     5941 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6001 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6061 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6121 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6181 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6241 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6301 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6361 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6421 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6481 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6541 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6601 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6661 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6721 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     6781 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     6841 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     6901 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     6961 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat at
//
