LOCUS       sequence-431530-        3842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) Stop; IRES2; Start; PuroR; Stop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3842)
  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 3842)
  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: AA333
FEATURES             Location/Qualifiers
     source          1..3842
                     /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"
     misc_feature    join(470..1044,1045..1047,1048..1056)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(637..654)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     864..883
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1072..1671
                     /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(1072..1091)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1568..1588
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1752..1768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1752..1768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1765..1787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1776..1792
                     /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(1800..1806,1807..1824,1825..1830))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1845..1866
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1983..2000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2154..2742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2234..2253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2920..3729)
                     /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     3643..3662
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3721..3740
                     /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 agctgaatgc atgagtaact gaggatccgc ccctctccct
      481 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
      541 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
      601 ctgtcgtctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
      661 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgacgacaa acaacgtctg
      721 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
      781 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
      841 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
      901 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtac acatgcttta
      961 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     1021 tcctttgaaa aacacgatga taatatggcc acaaccctgg aattcgccac catgaccgag
     1081 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     1141 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     1201 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     1261 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     1321 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     1381 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     1441 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     1501 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     1561 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     1621 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccta atgaattcca
     1681 gtcttcggaa ccaaatcgga tcccgggccc gtcgactgca gaggcctgca tgcaagcttg
     1741 gtgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     1801 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1861 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1921 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1981 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2041 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2101 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2161 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2221 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2281 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2341 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2401 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2461 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2521 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2581 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2641 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2701 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2761 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2821 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaagc
     2881 ccaatctgaa taatgttaca accaattaac caattctgat tagaaaaact catcgagcat
     2941 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     3001 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     3061 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     3121 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     3181 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     3241 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     3301 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     3361 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     3421 tgtttttccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     3481 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     3541 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     3601 cccatacaag cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     3661 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgacgttt cccgttgaat
     3721 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     3781 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacgg gccagagctg
     3841 ca
//
