LOCUS       sequence-307050-        7311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway cloning into P. tricornutum episomal vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7311)
  TITLE     Raffaela Abbriano Lab unpublished
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7311)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pPtPBR11_fcpShble_GWB
FEATURES             Location/Qualifiers
     source          1..7311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(515..889)
                     /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(1593..1611)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1635..1652)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1635..1651)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1644..1666)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     oriT            1928..2037
                     /note="incP origin of transfer"
     CDS             2070..2441
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     primer_bind     2656..2678
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2672..2863)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    3073..3197
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3238..3543)
                     /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     complement(3340..3359)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3885..4544)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4459..4478
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(4598..4604,4605..4622,4623..4628))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(4653..4777)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4806..4824)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4892..4996
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4997..5065,5066..5857)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5215..5234)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6028..6616
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6517..6536
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6770..6787
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        complement(6836..7022)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     misc_feature    join(7023..7311,1..215)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 cttctacaca gacaagatga aacaattcgg cattaatacc tgagagcagg aagagcaagg
       61 taaaaggtag tatttgttgg cgatccccct agagtctttt acatcttcgg aaaacaaaaa
      121 ctattttttc tttaatttct ttttttactt tctattttta atttatatat ttatattaaa
      181 aaatttaaat tataattatt tttatagcac gtgattgaag acgagctagt gttattcctg
      241 actgtgaaac caaagcggag tgactgcaac taatgaaaat taaattttta aggaaggata
      301 gagactccca actgttcgtg caccatgaga gcaaggatgc ccatttgtgc cgcacgtccc
      361 tggttgagtt cgatagcacg cttctgaagc ttcttgtcct cactgaaagt gtcccagcca
      421 aagtcgaggt agttgttgcg gccgcatggg cccagatctc cgaggcctcg gacccgtcgg
      481 gccgcgtcgg accggcggtg ttggtcggcg tcggtcagtc ctgctcctcg gccacgaagt
      541 gcacgcagtt gccggccggg tcgcgcaggg cgaactcccg cccccacggc tgctcgccga
      601 tctcggtcat ggccggcccg gaggcgtccc ggaagttcgt ggacacgacc tccgaccact
      661 cggcgtacag ctcgtccagg ccgcgcaccc acacccaggc cagggtgttg tccggcacca
      721 cctggtcctg gaccgcgctg atgaacaggg tcacgtcgtc ccggaccaca ccggcgaagt
      781 cgtcctccac gaagtcccgg gagaacccga gccggtcggt ccagaactcg accgctccgg
      841 cgacgtcgcg cgcggtgagc accggaacgg cactggtcaa cttggccatg ggtaccctca
      901 gctagaatat tatcgatacc gtcgacttgg ttaatttttc gatttcgaag atatgtgaag
      961 atccaggtgg tgtagatcac gaaagcgttc cgccgcgtgc caatccatcc atcggcaggg
     1021 atcgtcgttc aacctgccta cacgtgcttc gcacgtagaa attctaccag tacaacgaaa
     1081 gctgctgtcc gacgttacga acatggtcta gtcagacagt tgaaagaccc ggagttttga
     1141 gatcttcggt cgatcataaa aaaccttgct agcacagaaa tattgctgag ttacgttcac
     1201 accaaaaaca gagcactgtt ccaagtccgc aagttacttt tacatgtaaa agcgttgtct
     1261 atgtttatga aatcagcacc gcatgtcgaa aatgctattt tgagaatcgc cgatgctaat
     1321 cattggcctc taaaaaaacg gaagtatcaa gaattccaga caaatcagga tttacagata
     1381 acataaatcg cgaaatggaa ttttgcaacc aacgttaaca ggtatgtctc cttaccaaac
     1441 gctagtccag tccctcccag ctgatgtctt tcaaccaaca agtatctacg actgcagcac
     1501 taatgcaaat gataatataa aaaagaagac aatgtttttc ccagtgattc aactcgaatt
     1561 gcactaatcc tgttaggtac cagcttttgt tccctttagt gagggttaat ttcgagcttg
     1621 gcgtaatcat ggtcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     1681 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     1741 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcatcttccg
     1801 ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg
     1861 cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc
     1921 gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg
     1981 tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccggcta
     2041 ccgccggcgt aacagatgag ggcaagcgga tggctgatga aaccaagcca accaggaagg
     2101 gcagcccacc tatcaaggtg tactgccttc cagacgaacg aagagcgatt gaggaaaagg
     2161 cggcggcggc cggcatgagc ctgtcggcct acctgctggc cgtcggccag ggctacaaaa
     2221 tcacgggcgt cgtggactat gagcacgtcc gcgagctggc ccgcatcaat ggcgacctgg
     2281 gccgcctggg cggcctgctg aaactctggc tcaccgacga cccgcgcacg gcgcggttcg
     2341 gtgatgccac gatcctcgcc ctgctggcga agatcgaaga gaagcaggac gagcttggca
     2401 aggtcatgat gggcgtggtc cgcccgaggg cagagccatg acttttttag ccgctaaaac
     2461 ggccgggggg tgcgcgtgat tgccaagcac gtccccatgc gctccatcaa gaagagcgac
     2521 ttcgcggagc tggtgaagta catcaccgac gagcaaggca agacgatccg ccccgacacc
     2581 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     2641 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     2701 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     2761 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     2821 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     2881 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     2941 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     3001 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     3061 cgcaggtgat caaccacttt gtacaagaaa gctgaacgag aaacgtaaaa tgatataaat
     3121 atcaatatat taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa
     3181 catatccagt cactatggtc gacctgcaga ctggctgtgt ataagggagc ctgacattta
     3241 tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt ggctgagatc
     3301 agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg tcatcatgcg
     3361 ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt tatctgacag
     3421 cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact
     3481 ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa ccttaaactg
     3541 catttcacca gcccctgttc tcgtcagcaa aagagccgtt catttcaata aaccgggcga
     3601 cctcagccat cccttcctga ttttccgctt tccagcgttc ggcacgcaga cgacgggctt
     3661 cattctgcat ggttgtgctt accagaccgg agatattgac atcatatatg ccttgagcaa
     3721 ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc ttcatagcat acctcttttt
     3781 gacatacttc gggtatacat atcagtatat attcttatac cgcaaaaatc agcgcgcaaa
     3841 tacgcatact gttatctggc ttttagtaag ccggatccag atctttacgc cccgccctgc
     3901 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa
     3961 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
     4021 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa
     4081 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
     4141 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac
     4201 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg
     4261 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc
     4321 atacggaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa
     4381 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc
     4441 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
     4501 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct
     4561 cctgaaaatc tcgacggatc ctaactcaaa atccacacat tatacgagcc ggaagcataa
     4621 agtgtaaagc ctggggtgcc taatgcggcc gccatagtga ctggatatgt tgtgttttac
     4681 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat
     4741 tttacgtttc tcgttcagct tttttgtaca aacttgttga tcacctgctt cttgaagacg
     4801 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4861 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4921 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4981 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5041 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5101 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5161 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5221 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5281 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5341 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5401 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5461 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5521 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     5581 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5641 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5701 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5761 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5821 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5881 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5941 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6001 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6061 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6121 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     6181 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6241 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6301 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6361 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6421 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6481 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6541 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6601 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6661 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     6721 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     6781 gagcgaggaa gcggaagaga tgcctgcagg tcgactctag agtctgctct gtcatctttg
     6841 ccttcgttta tcttgcctgc tcatttttta gtatattctt cgaagaaatc acattacttt
     6901 atataatgta taattcatta tgtgataatg ccaatcgcta agaaaaaaaa agagtcatcc
     6961 gctaggtgga aaaaaaaaat gaaaatcatt accgaggcat aaaaaaatat agagtgtact
     7021 aggatcgctt gcctgtaact tacacgcgcc tcgtatcttt taatgatgga ataatttggg
     7081 aatttactct gtgtttattt atttttatgt tttgtatttg gattttagaa agtaaataaa
     7141 gaaggtagaa gagttacgga atgaagaaaa aaaaataaac aaaggtttaa aaaatttcaa
     7201 caaaaagcgt actttacata tatatttatt agacaagaaa agcagattaa atagatatac
     7261 attcgattaa cgataagtaa aatgtaaaat cacaggattt tcgtgtgtgg t
//
