LOCUS       sequence-431524-        3274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) Puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3274)
  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 3274)
  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: AA014
FEATURES             Location/Qualifiers
     source          1..3274
                     /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             501..1094
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     994..1014
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1184..1200)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1184..1200)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1197..1219)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1208..1224
                     /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(1232..1238,1239..1256,1257..1262))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1277..1298
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1415..1432)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1586..2174)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1666..1685)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2352..3161)
                     /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     3075..3094
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3153..3172
                     /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 accgagtaca agcccacggt gcgcctcgcc acccgcgacg
      541 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
      601 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
      661 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
      721 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
      781 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
      841 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc
      901 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
      961 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1021 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1081 gcaagcccgg tgccggatcc tgataaattc cagtcttcgg aaccaaatcg gatcccgggc
     1141 ccgtcgactg cagaggcctg catgcaagct tggtgtaatc atggtcatag ctgtttcctg
     1201 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     1261 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1321 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1381 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1441 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1501 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1561 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1621 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1681 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1741 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1801 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1861 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1921 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1981 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2041 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2101 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2161 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2221 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2281 ttttaaatta aaaatgaagt tttaaatcaa gcccaatctg aataatgtta caaccaatta
     2341 accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2401 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2461 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2521 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2581 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2641 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2701 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2761 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2821 gaatcaggat attcttctaa tacctggaat gctgtttttc cggggatcgc agtggtgagt
     2881 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2941 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     3001 tgtttcagaa acaactctgg cgcatcgggc ttcccataca agcgatagat tgtcgcacct
     3061 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     3121 tttaatcgcg gcctcgacgt ttcccgttga atatggctca taacacccct tgtattactg
     3181 tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa
     3241 catcagagat tttgagacac gggccagagc tgca
//
