LOCUS       sequence-431425-        3641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) Stop; IRES2; Start; BlastR; Stop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3641)
  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 3641)
  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: AA334
FEATURES             Location/Qualifiers
     source          1..3641
                     /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"
     regulatory      1066..1075
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1072..1470
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(1551..1567)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1551..1567)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1564..1586)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1575..1591
                     /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(1599..1605,1606..1623,1624..1629))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1644..1665
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1782..1799)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1953..2541)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2033..2052)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2719..3528)
                     /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     3442..3461
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3520..3539
                     /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 catggccaag
     1081 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     1141 cccatctctg aggactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     1201 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     1261 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     1321 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     1381 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     1441 ctgccctctg gttatgtgtg ggagggctaa tgaattccag tcttcggaac caaatcggat
     1501 cccgggcccg tcgactgcag aggcctgcat gcaagcttgg tgtaatcatg gtcatagctg
     1561 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     1621 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     1681 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1741 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     1801 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1861 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1921 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     1981 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2041 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2101 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2161 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2221 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2281 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2341 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2401 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2461 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     2521 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     2581 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2641 tagatccttt taaattaaaa atgaagtttt aaatcaagcc caatctgaat aatgttacaa
     2701 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     2761 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     2821 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     2881 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     2941 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     3001 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     3061 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     3121 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     3181 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gtttttccgg ggatcgcagt
     3241 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     3301 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     3361 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaagc gatagattgt
     3421 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     3481 gttggaattt aatcgcggcc tcgacgtttc ccgttgaata tggctcataa caccccttgt
     3541 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc
     3601 aatgtaacat cagagatttt gagacacggg ccagagctgc a
//
