LOCUS       sequence-473907-        3645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter codon optimized for Dictyostelid species.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3645)
  TITLE     Selection-free CRISPR-Cas9 editing protocol for distant Dictyostelid
            species
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3645)
  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            "Unpublished"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: pMG005_mNeonGreen_P2A
FEATURES             Location/Qualifiers
     source          1..3645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     278..300
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     292..309
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             396..416
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             468..515
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             516..581
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1287..1313
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1326..1382
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(1788..1805)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1898..2486)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1978..1997)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2657..3448,3449..3517))
                     /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     3280..3299
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3518..3622)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca
      361 aggccgcata aaaaatggtg agttcattaa gaccaccaaa gaaaaagaga aaggtttgtc
      421 caggtgatag atggagtagt acaggaggtg gaagaagtag aacttcaaag agacctgccg
      481 ctacaaaaaa ggccggacaa gcaaagaaga aaaaggacta caaagatcac gatggagatt
      541 ataaggatca cgatatagat tacaaagatg atgatgataa agtatctaag ggagaagaag
      601 ataatatggc ctctcttcct gccacccatg aattacacat tttcggttct attaatggag
      661 ttgatttcga tatggttgga caaggaactg gtaatccaaa tgatggttat gaggaattaa
      721 atcttaaaag tacaaaggga gatcttcaat ttagtccatg gatcttagta cctcacatcg
      781 gatacggttt tcaccaatac ttgccatacc cagatggtat gagtccattt caagccgcta
      841 tggtagatgg aagtggttac caagtacacc gtacaatgca atttgaagat ggagctagtc
      901 ttacagttaa ttaccgttat acatacgagg gatctcatat caaaggtgag gcccaagtaa
      961 agggaaccgg tttccctgca gatggaccag taatgactaa tagtttaact gccgccgatt
     1021 ggtgtagatc aaaaaagaca tatccaaatg ataagactat aatatcaaca ttcaaatggt
     1081 cttacactac tggaaatggt aagagatatc gttctaccgc cagaacaact tacacatttg
     1141 ccaagccaat ggcagcaaat tacttaaaga atcaacctat gtacgttttt cgtaaaacag
     1201 agttgaagca ctcaaagacc gagttgaatt ttaaagagtg gcaaaaggca tttacagatg
     1261 ttatgggaat ggatgaatta tataagtacc catacgatgt acctgattat gctggtggtt
     1321 ctggtgctac aaatttctct ttgttaaagc aagctggaga tgttgaagaa aatcctggac
     1381 cttctagtgg acctagtgga tctagttctt gaattattta ataaataata aaaaaacaaa
     1441 ttgttgtaat aatctaatat tttctttttt ttttaatttt ttttttttaa atcttaataa
     1501 ttattaagtt attttaattt tttttttttt tttttttttt tttttttttt ttttctatca
     1561 aaaaaatcaa atatatttaa aaaatttatt atttacagat acattttgaa tggtgaagat
     1621 aaatatatgc attagatgta aaacagccaa agagtatgaa aatcaaaaag atactgggcc
     1681 tcatgggcct tccgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1741 taacatggtc atagctgttt ccttgcgtat tgggcgctct ccgcttcctc gctcactgac
     1801 tcgctgcgct cggtcgttcg ggtaaagcct ggggtgccta atgagcaaaa ggccagcaaa
     1861 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1921 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1981 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2041 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2101 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2161 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2221 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2281 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2341 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2401 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2461 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2521 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2581 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2641 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2701 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2761 gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca ccggctccag
     2821 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2881 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2941 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3001 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3061 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3121 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3181 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3241 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3301 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3361 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3421 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3481 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3541 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3601 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
