LOCUS       sequence-200867-        4510 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses mCherry under endogenous actin promoter. It can be used to
            transfect Corallochytrium limacisporum (Corallochytrea) cells and 
            visualize them by fluorescent microscope. Puromycin resistant..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4510)
  AUTHORS   Kozyczkowska A, Najle SR, Ocana-Pallares E, Areste C, Shabardina V, 
            Ara PS, Ruiz-Trillo I, Casacuberta E
  TITLE     Stable transfection in protist Corallochytrium limacisporum 
            identifies novel cellular features among unicellular animals 
            relatives.
  JOURNAL   Curr Biol. 2021 Sep 27;31(18):4104-4110.e5. doi: 
            10.1016/j.cub.2021.06.061. Epub 2021 Jul 21.
  PUBMED    34293333
REFERENCE   2  (bases 1 to 4510)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cub.2021.06.061"; journalName: "Curr Biol"; date: 
            "2021-09-27- 27"; volume: "31"; issue: "18"; pages: "4104-4110.e5"
COMMENT     Sequence Label: Clim Actin MCH Puro (CAMP)
FEATURES             Location/Qualifiers
     source          1..4510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    104..125
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(140..145,146..163,164..170)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    178..194
                     /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)."
     primer_bind     183..205
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     202..218
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     202..218
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             808..1515
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGAHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(953..971)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1221..1240
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1415..1438
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1525..2124
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1525..1544)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2021..2041
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2559..2578
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2707..2775,2776..3567)
                     /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     complement(2925..2944)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3738..4326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4227..4246
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4480..4497
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
       61 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
      121 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      181 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgaaa
      241 tgctgggcgc cttgaaactg aacctgttcg gatccgggtt gcgcgcttcc attgttttgg
      301 tttccgtgtc tgcaaggcgg gtctggacgt gacgctttgg gggagaggga gagcagagat
      361 acaattctag ccacgaattt cagccaatag tcacagaggg gagttgcgat cctgcgaatt
      421 ggcttaggtc tgtgaggtct ttcacggttt cgaaagcagc gcagagggga gaccgactac
      481 caaaagccat aacactccac cgagatatcc gcactcttcg ctagatgagc ttgcttctcc
      541 tcttgtcaca aatcagccca atgatagtca ctgttcaaca acggtgcagt gtaaatattc
      601 gttgtgcgtc cttgatgagc agaaggtacg agaagcagga aatcatttca taatggaaaa
      661 ccattcactg tctttctgta ttgcgaaaag agtgatcata ggtaatcgca acggcgggtc
      721 tttagaacag tcacatacca gatcatgggc atggagacag tagagcaact acgcaagaca
      781 actattgtcc ccgggactag tgatatcatg gtgagcaagg gcgaggagga caacatggcc
      841 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
      901 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
      961 aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     1021 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     1081 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1141 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1201 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1261 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1321 ctgaaggacg gcgcccacta cgacgccgag gtcaagacca cctacaaggc caagaagccc
     1381 gtgcagctgc ccggcgccta caacgtcaac atcaagctgg acatcacctc ccacaacgag
     1441 gactacacca tcgtggaaca gtacgagcgc gccgagggcc gccactccac cggcggcatg
     1501 gacgagctgt acaaggcggc cgccatgacc gagtacaagc ccacggtgcg cctcgccacc
     1561 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     1621 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     1681 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     1741 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     1801 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     1861 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     1921 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1981 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     2041 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     2101 atgacccgca agcccggtgc ctgacctcga gtaaacaaat gttttgttgg aagatttgat
     2161 ttgtcttttt tggagtgctt agttagtgca ctcctagatg agcaaacatt accacagtgt
     2221 atcgcagcat aaaataaaat tataaaatgt ctttgtagat caaaagcatt acaaacaaat
     2281 atttaaactt tgatcacttt tttcaaattc gtcaatatga ctaattaaca tctgggttat
     2341 agatgacaag catgaatgtt tcatttattt taaaaaaaaa ataaataagt attttttcat
     2401 ttgcatcacc atcaaaaata tttccattga aaagtcatcg actggctatt taaatttcta
     2461 gagggcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     2521 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     2581 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     2641 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttgaaaaagg
     2701 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2761 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2821 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2881 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2941 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3001 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3061 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3121 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3181 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3241 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3301 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3361 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3421 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3481 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3541 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3601 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3661 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3721 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3781 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3841 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     3901 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3961 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4021 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4081 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4141 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4201 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4261 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4321 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4381 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4441 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4501 gcggaagagc
//
