LOCUS       sequence-183791-        7012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN Backbone for Phaeodactylum tricornutum_ NR promotor_HD.
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_HD
FEATURES             Location/Qualifiers
     source          1..7012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        500..518
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      520..529
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             529..594
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             601..621
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        join(1090..1095,1096..1113,1114..1120)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1174..1833
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1240..1259)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2175..2480
                     /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     2359..2378
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2784..3371
                     /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(3389..3406)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             3916..4485
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4739..4757)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4844..4861)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5015..5603)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5095..5114)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5774..6565,6566..6634))
                     /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     6397..6416
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6635..6739)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6807..6825
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6863..6885)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6985..7004
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcaccatatg cggaagtgac tgtaaacgag aagtgcacga agccttttct tgtgacgtca
       61 caaaccgaac agccctacgt ggcgtgcgac tttccggcac tttggatatc gttgccctat
      121 atgtattggg tgtatacatg tcgtattcca actacgacag aaaacagtca gttacgggta
      181 ttcgaacttc cggcacctgc agcgaggagt tttgtgtccg tcgcacatcc tccgtgcttt
      241 cggcacctac gtaggcggca aacttcctgc cttccgcccg ccttgcggca ttccggctcc
      301 gggccagaat tgcccgggtg ttcacaattt tgcctcctca cgaaaaaacg ttctactttg
      361 tattttggtc gggtttcgga tccttccagc aactattctc attcaaagtc accacttgtg
      421 cgaacggaat tcgatatcat cgactaattc gagctctctg gctaactaga gaacccactg
      481 cttactggct tatcgaaatt aatacgactc actatagggg ccaccatgga ctataaggac
      541 cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc
      601 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgtaga tttgagaact
      661 ttgggatatt cacagcagca gcaggaaaag atcaagccca aagtgaggtc gacagtcgcg
      721 cagcatcacg aagcgctggt gggtcatggg tttacacatg cccacatcgt agccttgtcg
      781 cagcaccctg cagcccttgg cacggtcgcc gtcaagtacc aggacatgat tgcggcgttg
      841 ccggaagcca cacatgaggc gatcgtcggt gtggggaaac agtggagcgg agcccgagcg
      901 cttgaggccc tgttgacggt cgcgggagag ctgagagggc ctccccttca gctggacacg
      961 ggccagttgc tgaagatcgc gaagcgggga ggagtcacgg cggtcgaggc ggtgcacgcg
     1021 tggcgcaatg cgctcacggg agcacccctc aacctgacag agaccgcggc cgcattaggc
     1081 accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat
     1141 ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     1201 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     1261 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     1321 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     1381 catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     1441 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     1501 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     1561 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     1621 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     1681 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     1741 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     1801 cagtactgcg atgagtggca gggcggggcg taaagatctg gatccggctt actaaaagcc
     1861 agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg
     1921 tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg
     1981 acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag
     2041 cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca
     2101 ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa
     2161 caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc
     2221 tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc
     2281 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
     2341 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta
     2401 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
     2461 tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac
     2521 ggtctcgact cacgcctgag caggtagtgg ctattgcatc ccatgacggg ggcagacccg
     2581 cactggagtc aatcgtggcc cagctttcga ggccggaccc cgcgctggcc gcactcacta
     2641 atgatcatct tgtagcgctg gcctgcctcg gcggacgacc cgccttggat gcggtgaaga
     2701 aggggctccc gcacgcgcct gcattgatta agcggaccaa cagaaggatt cccgagagga
     2761 catcacatcg agtggcaggt tcccaactcg tgaagagtga acttgaggag aaaaagtcgg
     2821 agctgcggca caaattgaaa tacgtaccgc atgaatacat cgaacttatc gaaattgcta
     2881 ggaactcgac tcaagacaga atccttgaga tgaaggtaat ggagttcttt atgaaggttt
     2941 atggataccg agggaagcat ctcggtggat cacgaaaacc cgacggagca atctatacgg
     3001 tggggagccc gattgattac ggagtgatcg tcgacacgaa agcctacagc ggtgggtaca
     3061 atcttcccat cgggcaggca gatgagatgc aacgttatgt cgaagaaaat cagaccagga
     3121 acaaacacat caatccaaat gagtggtgga aagtgtatcc ttcatcagtg accgagttta
     3181 agtttttgtt tgtctctggg catttcaaag gcaactataa ggcccagctc acacggttga
     3241 atcacattac gaactgcaat ggtgcggttt tgtccgtaga ggaactgctc attggtggag
     3301 aaatgatcaa agcgggaact ctgacactgg aagaagtcag acgcaagttt aacaatggcg
     3361 agatcaattt ccgctcataa aaaatcagcc tcgactgtgc cttctagttg ccagccatct
     3421 gttgtttgcc cctcccccgt gccttccttg accctggaag cttcaattta ctttaagact
     3481 agcgcaattt ttttcttaca ttagatgaac ataaaacgac gatgaggatc actgttaggg
     3541 aaacgatgaa ctacgctgag gacttcctat tattcgtcct ttgatggcgt ggttgttctt
     3601 tggtttgcgg ccgagcgcaa cgaccaagcc ctgtaccaaa aacgaagttg ataatcagat
     3661 atttgagttg taacaagaaa ctaaagcatt attttagcag tagcacgaat caggatctaa
     3721 aatgaacgca tccggagaga ttcaaaattc aatctgtttg gacctggata agacacaaga
     3781 gcgacatcct gacatgaacg ccgtaaacag caaatcctgg ttgaacacgt atccttttgg
     3841 gggcctcccg ctacgacgct cgctccagct ggggcttcct tactatacac agcgcgcata
     3901 tttcacggtt gccagatgac cactcttgac gacacggctt accggtaccg caccagtgtc
     3961 ccgggggacg ccgaggccat cgaggcactg gatgggtcct tcaccaccga caccgtcttc
     4021 cgcgtcaccg ccaccgggga cggcttcacc ctgcgggagg tgccggtgga cccgcccctg
     4081 accaaggtgt tccccgacga cgaatcggac gacgaatcgg acgacgggga ggacggcgac
     4141 ccggactccc ggacgttcgt cgcgtacggg gacgacggcg acctggcggg cttcgtggtc
     4201 gtctcgtact ccggctggaa ccgccggctg accgtcgagg acatcgaggt cgccccggag
     4261 caccgggggc acggggtcgg gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag
     4321 cggggcgccg ggcacctctg gctggaggtc accaacgtca acgcaccggc gatccacgcg
     4381 taccggcgga tggggttcac cctctgcggc ctggacaccg ccctgtacga cggcaccgcc
     4441 tcggacggcg agcaggcgct ctacatgagc atgccctgcc cctgaaaacc ttccttaaaa
     4501 atttaatttt cattagttgc agtcactccg ctttggtttc acagtcagga ataacactag
     4561 ctcgtcttca ccatggatgc caatctcgcc tattcatggt gtataaaagt tcaacatcca
     4621 aagctagaac ttttggaaag agaaagaata tccgaatagg gcacggcgtg ccgtattgtt
     4681 ggagtggact agcagaaagt gaggaaggca caggatgagt tttctcgagg ccggactccc
     4741 tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga atcggccaac
     4801 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4861 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4921 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4981 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5041 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5101 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5161 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5221 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5281 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5341 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5401 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5461 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5521 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5581 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5641 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5701 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5761 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5821 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5881 taccatctgg ccccagtgct gcaatgatac cgcgggaccc acgctcaccg gctccagatt
     5941 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6001 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6061 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6121 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6181 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6241 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6301 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6361 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6421 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6481 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6541 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6601 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6661 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6721 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     6781 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     6841 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     6901 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     6961 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gt
//
