LOCUS       sequence-183794-        7058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN Backbone for Phaeodactylum tricornutum_ fcpA promotor_HD.
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_HD
FEATURES             Location/Qualifiers
     source          1..7058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..46)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(200..788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(280..299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(959..1750,1751..1819))
                     /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     1582..1601
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1820..1924)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1992..2010
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2048..2070)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2170..2189
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2270..2288
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2270..2287
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        2816..2834
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2836..2845
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2845..2910
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2917..2937
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        join(3406..3411,3412..3429,3430..3436)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3490..4149
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3556..3575)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4491..4796
                     /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     4675..4694
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             5100..5687
                     /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(5705..5722)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             6356..6730
                     /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(6982..7000)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
       61 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
      121 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
      181 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      241 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      301 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      361 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      421 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      481 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      541 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      601 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
      661 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      721 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      781 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      841 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
      901 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
      961 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     1021 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     1081 gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc
     1141 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1201 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1261 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1321 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1381 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     1441 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     1501 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     1561 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     1621 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     1681 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     1741 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     1801 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     1861 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     1921 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     1981 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     2041 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     2101 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     2161 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt
     2221 tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac
     2281 actatagaac cagatccccc gggctgcagg acgcaatgga ggattatcac cgcaaaaatg
     2341 aacttcgaaa aaaactttcg agcgaccatg gaaaaggagg atcagattca gattacaaca
     2401 gtggattgct ctggtagcaa atatcttctg ctagattggc tcatggtcgg ttttggacgt
     2461 tcgaagctca ccgtcaaaag aaacaaaaga gaagaatgac gtcttcgtga cgtagaatct
     2521 acgactgtac tcggatctgg gaaatgaatt gactcacggt cttcttcgag tcctgttaca
     2581 ggcccttggt ccgaaccccc acacgatttt tgcaccaaag atttgcttca atttgctgga
     2641 tgttttgact gcaagatcag ctggcctagc aagagtgctc gtgttgcttc gtcgggaatc
     2701 cctacgaatt tcagttctgc acaaatttgt ctgccgtttc gagaattcga tatcatcgac
     2761 taattcgagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata
     2821 cgactcacta taggggccac catggactat aaggaccacg acggagacta caaggatcat
     2881 gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt
     2941 atccacggag tcccagcagc cgtagatttg agaactttgg gatattcaca gcagcagcag
     3001 gaaaagatca agcccaaagt gaggtcgaca gtcgcgcagc atcacgaagc gctggtgggt
     3061 catgggttta cacatgccca catcgtagcc ttgtcgcagc accctgcagc ccttggcacg
     3121 gtcgccgtca agtaccagga catgattgcg gcgttgccgg aagccacaca tgaggcgatc
     3181 gtcggtgtgg ggaaacagtg gagcggagcc cgagcgcttg aggccctgtt gacggtcgcg
     3241 ggagagctga gagggcctcc ccttcagctg gacacgggcc agttgctgaa gatcgcgaag
     3301 cggggaggag tcacggcggt cgaggcggtg cacgcgtggc gcaatgcgct cacgggagca
     3361 cccctcaacc tgacagagac cgcggccgca ttaggcaccc caggctttac actttatgct
     3421 tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc aggagctaag
     3481 gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat
     3541 cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt
     3601 cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg
     3661 gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg
     3721 aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag
     3781 caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta
     3841 cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg
     3901 tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat
     3961 ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat
     4021 acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat
     4081 ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc
     4141 ggggcgtaaa gatctggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc
     4201 gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga
     4261 ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca
     4321 aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg
     4381 tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg
     4441 gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa atgcagttta
     4501 aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata
     4561 ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag
     4621 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga
     4681 tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca
     4741 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt
     4801 caggctccct tatacacagc cagtctgcag gtcgacggtc tcgactcacg cctgagcagg
     4861 tagtggctat tgcatcccat gacgggggca gacccgcact ggagtcaatc gtggcccagc
     4921 tttcgaggcc ggaccccgcg ctggccgcac tcactaatga tcatcttgta gcgctggcct
     4981 gcctcggcgg acgacccgcc ttggatgcgg tgaagaaggg gctcccgcac gcgcctgcat
     5041 tgattaagcg gaccaacaga aggattcccg agaggacatc acatcgagtg gcaggttccc
     5101 aactcgtgaa gagtgaactt gaggagaaaa agtcggagct gcggcacaaa ttgaaatacg
     5161 taccgcatga atacatcgaa cttatcgaaa ttgctaggaa ctcgactcaa gacagaatcc
     5221 ttgagatgaa ggtaatggag ttctttatga aggtttatgg ataccgaggg aagcatctcg
     5281 gtggatcacg aaaacccgac ggagcaatct atacggtggg gagcccgatt gattacggag
     5341 tgatcgtcga cacgaaagcc tacagcggtg ggtacaatct tcccatcggg caggcagatg
     5401 agatgcaacg ttatgtcgaa gaaaatcaga ccaggaacaa acacatcaat ccaaatgagt
     5461 ggtggaaagt gtatccttca tcagtgaccg agtttaagtt tttgtttgtc tctgggcatt
     5521 tcaaaggcaa ctataaggcc cagctcacac ggttgaatca cattacgaac tgcaatggtg
     5581 cggttttgtc cgtagaggaa ctgctcattg gtggagaaat gatcaaagcg ggaactctga
     5641 cactggaaga agtcagacgc aagtttaaca atggcgagat caatttccgc tcataaaaaa
     5701 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5761 tccttgaccc tggaagcttc agaagcgtgc tatcgaactc aaccagggac gtgcggcaca
     5821 aatgggcatc cttgctctca tggtgcacga acagttggga gtctctatcc ttccttaaaa
     5881 atttaatttt cattagttgc agtcactccg ctttggtttc acagtcagga ataacactag
     5941 ctcgtcttca ccatggatgc caatctcgcc tattcatggt gtataaaagt tcaacatcca
     6001 aagctagaac ttttggaaag agaaagaata tccgaatagg gcacggcgtg ccgtattgtt
     6061 ggagtggact agcagaaagt gaggaaggca caggatgagt tttctcgaga cataccttca
     6121 gcgtcgtctt cactgtcaca gtcaactgac agtaatcgtt gatccggaga gattcaaaat
     6181 tcaatctgtt tggacctgga taagacacaa gagcgacatc ctgacatgaa cgccgtaaac
     6241 agcaaatcct ggttgaacac gtatcctttt gggggcctcc cgctacgacg ctcgctccag
     6301 ctggggcttc cttactatac acagcgcgca tatttcacgg ttgccagatg tcaagatggc
     6361 caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt
     6421 ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt
     6481 ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac
     6541 cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt
     6601 gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg
     6661 ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga
     6721 gcaggactga accttcctta aaaatttaat tttcattagt tgcagtcact ccgctttggt
     6781 ttcacagtca ggaataacac tagctcgtct tcaccatgga tgccaatctc gcctattcat
     6841 ggtgtataaa agttcaacat ccaaagctag aacttttgga aagagaaaga atatccgaat
     6901 agggcacggc gtgccgtatt gttggagtgg actagcagaa agtgaggaag gcacaggatg
     6961 agttttctcg aggccggact ccctatagtg agtcgtatta atttcgataa gccaggttaa
     7021 cctgcattaa tgaatcggcc aacgcgcggg gagaggcg
//
