LOCUS       sequence-434143-        4268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) Stop; IRES2; Start; HygroR; Stop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4268)
  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 4268)
  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: AA335
FEATURES             Location/Qualifiers
     source          1..4268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(604..1413)
                     /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     1327..1346
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1405..1424
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1555..1577)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1677..1696
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1890..1912
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    join(1996..2570,2571..2573,2574..2582)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2163..2180)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2390..2409
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2598..3623
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(3704..3720)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3704..3720)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3717..3739)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3728..3744
                     /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(3752..3758,3759..3776,3777..3782))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3797..3818
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3935..3952)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4106..4268,1..426))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4186..4205)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
       61 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
      121 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
      181 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
      241 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
      301 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
      361 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
      421 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
      481 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
      541 taaaaatgaa gttttaaatc aagcccaatc tgaataatgt tacaaccaat taaccaattc
      601 tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
      661 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
      721 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
      781 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
      841 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
      901 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
      961 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     1021 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     1081 atattcttct aatacctgga atgctgtttt tccggggatc gcagtggtga gtaaccatgc
     1141 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     1201 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     1261 aaacaactct ggcgcatcgg gcttcccata caagcgatag attgtcgcac ctgattgccc
     1321 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     1381 cggcctcgac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     1441 agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag
     1501 attttgagac acgggccaga gctgcatcgc gcgtttcggt gatgacggtg aaaacctctg
     1561 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     1621 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     1681 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     1741 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     1801 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     1861 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     1921 agaattcgag ctcggtacct cgcgaataca tctagatgaa gacggaagct gaatgcatga
     1981 gtaactgagg atccgcccct ctccctcccc cccccctaac gttactggcc gaagccgctt
     2041 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg
     2101 caatgtgagg gcccggaaac ctggccctgt cgtcttgacg agcattccta ggggtctttc
     2161 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga
     2221 agcttcttga cgacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc
     2281 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc
     2341 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc
     2401 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga
     2461 tctggggcct cggtacacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg
     2521 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa
     2581 ccctggaatt cgccaccatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     2641 tgatcgaaaa gttcgacagc gtgtccgacc tgatgcagct ctcggagggc gaagaatctc
     2701 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     2761 atggtttcta caaagatcgt tatgttcatc ggcactttgc atcggccgcg ctcccgattc
     2821 cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc tcccgccgtg
     2881 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     2941 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     3001 cattcggacc gcaaggaatc ggtcaataca ctacgtggcg tgatttcata tgcgcgattg
     3061 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     3121 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     3181 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     3241 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     3301 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     3361 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     3421 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     3481 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     3541 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     3601 ctcgtccgag ggcaaaggaa taatgaattc cagtcttcgg aaccaaatcg gatcccgggc
     3661 ccgtcgactg cagaggcctg catgcaagct tggtgtaatc atggtcatag ctgtttcctg
     3721 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3781 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3841 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3901 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3961 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4021 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4081 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4141 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4201 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4261 tgtccgcc
//
