LOCUS       sequence-431535-        3385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) TagRFP657.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3385)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 3385)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA267
FEATURES             Location/Qualifiers
     source          1..3385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             444..500
                     /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"
     CDS             513..1214
                     /codon_start=1
                     /product="far-red monomeric fluorescent protein with an 
                     emission peak at 657 nm (Morozova et al., 2010)"
                     /label=TagRFP657
                     /note="derived from mKate"
                     /translation="MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV
                     VEGGPLPFAFDILATSFMYGSHTFINHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPADGGLEGRTALALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDYRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1295..1311)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1295..1311)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1308..1330)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1319..1335
                     /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(1343..1349,1350..1367,1368..1373))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1388..1409
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1526..1543)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1697..2285)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1777..1796)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2463..3272)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3186..3205
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3264..3283
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagacgga agcgcaacaa acttctctct gctgaaacaa gccggagatg
      481 tcgaagagaa tcctggaccg gcaccgggat ccatgagcga gctgatcacc gagaacatgc
      541 acatgaagct gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg
      601 gcgaaggcaa gccctacgag ggcacccaga cccagagaat caaggtggtc gagggcggcc
      661 ctctcccctt cgccttcgac atcctggcta ccagcttcat gtacggcagt cacaccttca
      721 tcaaccacac ccagggcatc cccgacttct ggaagcagtc cttccctgag ggcttcacat
      781 gggagagagt caccacatac gaggacgggg gcgtgctgac cgctacccag gacaccagcc
      841 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc ccatccaacg
      901 gccctgtgat gcagaagaaa acactcggct gggaggccca caccgagatg ctgtaccccg
      961 ctgacggcgg cctggaaggc agaaccgcgc tggccctgaa gctcgtgggc gggggccacc
     1021 tgatctgcaa cttcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc
     1081 ccggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccgac aaagagacct
     1141 acgtcgagca gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc
     1201 acaagcttaa ttaatgaatt ccagtcttcg gaaccaaatc ggatcccggg cccgtcgact
     1261 gcagaggcct gcatgcaagc ttggtgtaat catggtcata gctgtttcct gtgtgaaatt
     1321 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1381 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1441 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1501 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1561 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1621 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1681 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1741 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1801 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1861 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1921 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1981 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2041 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2101 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2161 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2221 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2281 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2341 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2401 aaaaatgaag ttttaaatca agcccaatct gaataatgtt acaaccaatt aaccaattct
     2461 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2521 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2581 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2641 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2701 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2761 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2821 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2881 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2941 tattcttcta atacctggaa tgctgttttt ccggggatcg cagtggtgag taaccatgca
     3001 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3061 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3121 aacaactctg gcgcatcggg cttcccatac aagcgataga ttgtcgcacc tgattgcccg
     3181 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3241 ggcctcgacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     3301 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     3361 ttttgagaca cgggccagag ctgca
//
