LOCUS       sequence-195301-        4838 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Golden-Gate compatible fungal transformation vector carrying 
            Hygromycin Resistance with RFP screening cassette (RFP knocked out 
            by GG reaction, allowing for red-white screening).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4838)
  AUTHORS   Pennington HG, Youles M, Kamoun S
  TITLE     Golden-Gate compatible Magnaporthe oryzae protoplast transformation 
            vectors.
  JOURNAL   Figshare (2017)
REFERENCE   2  (bases 1 to 4838)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Figshare 
            (2017)"
COMMENT     Sequence Label: pCB1532H-RFP
FEATURES             Location/Qualifiers
     source          1..4838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             495..506
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    524..540
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(548..554,555..572,573..578))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    593..614
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(758..778)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(758..776)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(797..813)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(797..813)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    821..837
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(845..851,852..869,870..875))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    890..911
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1028..1045)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1199..1787)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1279..1298)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1958..2749,2750..2818))
                     /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     2581..2600
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3211..4236
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANILFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     4386..4408
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4400..4417
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4401..4417
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4427..4446
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4427..4445
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(4490..4517)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(4533..4604)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(4649..4838,1..488))
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
ORIGIN
        1 attttgattt cacctttcag agcaccgtcc tccgggtaca tacgttcggt ggaagcttcc
       61 caacccatgg tttttttctg cataaccgga ccgtcggacg ggaagttggt accacgcagt
      121 ttaactttgt agatgaactc accgtcttgc agggaggagt cctgggtaac ggtaacaaca
      181 ccaccgtcct cgaagttcat aacacgttcc catttgaaac cttccgggaa ggacagtttc
      241 aggtagtccg ggatgtcagc cgggtgttta acgtaagctt tggaaccgta ctggaactgc
      301 ggggacagga tgtcccaagc gaacggcagc ggaccacctt tggtaacttt cagtttagcg
      361 gtctgggtac cttcgtacgg acgaccttca ccttcacctt cgatttcgaa ctcgtgaccg
      421 ttaacggaac cttccatacg aactttgaaa cgcatgaact ctttgataac gtcctcggag
      481 gaagccatct agtatttctc ctctttctct agtatgtgtg aaattgttat ccgctcacaa
      541 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      601 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
      661 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgatatcg
      721 gtctcactcc accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc
      781 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
      841 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
      901 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
      961 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1021 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1081 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1141 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1201 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1261 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1321 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1381 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1441 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1501 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1561 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1621 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1681 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1741 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1801 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1861 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     1921 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     1981 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2041 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgtg
     2101 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2161 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2221 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     2281 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2341 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2401 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2461 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2521 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     2581 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2641 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     2701 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     2761 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     2821 tcttcctttt tcaatgggtg atcgacgtta actgatattg aaggagcatt ttttgggctt
     2881 ggctggagct agtggaggtc aacaatgaat gcctattttg gtttagtcgt ccaggcggtg
     2941 agcacaaaat ttgtgtcgtt tgacaagatg gttcatttag gcaactggtc agatcagccc
     3001 cacttgtagc agtagcggcg gcgctcgaag tgtgactctt attagcagac aggaacgagg
     3061 acattattat catctgctgc ttggtgcacg ataacttggt gcgtttgtca agcaaggtaa
     3121 gtggacgacc cggtcatacc ttcttaagtt cgcccttcct ccctttattt cagattcaat
     3181 ctgacttacc tattctaccc aagcatccaa atgaaaaagc ctgaactcac cgcgacgtct
     3241 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     3301 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     3361 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     3421 gcgctcccga ttccggaagt gcttgacatt ggggagttca gcgagagcct gacctattgc
     3481 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     3541 gttctccagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     3601 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     3661 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     3721 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     3781 gtccggcacc tcgtgcatgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     3841 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3901 aacatcctct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3961 cggaggcatc cggagcttgc aggatcgccg cgcctccggg cgtatatgct ccgcattggt
     4021 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     4081 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     4141 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     4201 cgacgcccca gcactcgtcc gagggcaaag gaatagcgag cccattaacg cttacaattt
     4261 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     4321 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     4381 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     4441 atagggcgaa ttgggtaccg ggccccccct cgagcgtgag acctataaac gcagaaaggc
     4501 ccacccgaag gtgagccagt gtgactctag tagagagcgt tcaccgacaa acaacagata
     4561 aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct ggctctagta
     4621 gcgatctaca ctagcactat cagcgttatt aagcaccggt ggagtgacga ccttcagcac
     4681 gttcgtactg ttcaacgatg gtgtagtcct cgttgtggga ggtgatgtcc agtttgatgt
     4741 cggttttgta agcacccggc agctgaaccg gttttttagc catgtaggtg gttttaactt
     4801 cagcgtcgta gtgaccaccg tctttcagtt tcagacgc
//
