LOCUS       sequence-431477-        3208 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (Selection) puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3208)
  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 3208)
  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: AA068
FEATURES             Location/Qualifiers
     source          1..3208
                     /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             450..1046
                     /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(450..469)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     946..966
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1118..1134)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1118..1134)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1131..1153)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1142..1158
                     /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(1166..1172,1173..1190,1191..1196))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1211..1232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1349..1366)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1520..2108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1600..1619)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2286..3095)
                     /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     3009..3028
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3087..3106
                     /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 tgaagacgta ggagccacca tgaccgagta caagcccacg gtgcgcctcg
      481 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
      541 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
      601 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
      661 ccgccgtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
      721 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
      781 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct
      841 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
      901 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
      961 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     1021 ggtgcatgac ccgcaagccc ggtgccggaa gcacgtcttc atcggatccc gggcccgtcg
     1081 actgcagagg cctgcatgca agcttggtgt aatcatggtc atagctgttt cctgtgtgaa
     1141 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1201 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1261 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1321 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1381 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1441 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1501 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1561 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1621 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1681 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1741 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1801 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1861 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     1921 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     1981 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2041 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2101 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2161 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2221 attaaaaatg aagttttaaa tcaagcccaa tctgaataat gttacaacca attaaccaat
     2281 tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2341 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2401 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2461 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2521 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2581 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2641 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2701 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2761 ggatattctt ctaatacctg gaatgctgtt tttccgggga tcgcagtggt gagtaaccat
     2821 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2881 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2941 agaaacaact ctggcgcatc gggcttccca tacaagcgat agattgtcgc acctgattgc
     3001 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3061 cgcggcctcg acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     3121 taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag
     3181 agattttgag acacgggcca gagctgca
//
