LOCUS       sequence-431456-        3232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (N' terminus) luminescent reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3232)
  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 3232)
  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: AA416
FEATURES             Location/Qualifiers
     source          1..3232
                     /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"
     CDS             complement(250..1146)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHAGLCSRRARDPMKTAASLSCSLLSCSFLCSSFSTRR
                     QNAFAQPPAGHSVDGYSEQQGAVGVDQALVDNFVAVPQGPCYSDLFAVEHGDAFIRPSE
                     IVDHVRRNPVDYQCAIVQDHLKVMIIHRVHHLKNFFDLAHLVAAQTFIRDDDMDVDLQP
                     IFTAQDNPLDRSYGHPEILKQTGHTSLFKDLVQVVAGCLSPVPNEIFECKDHRRIETEE
                     RVRDRLTHGGGSLLRLHLDVFARYRARILWPSFYNVVTGKTLALPNLIALQHIPLSPAG
                     VIAKRPAPIALPNSCAA"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     regulatory      451..460
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             460..501
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             502..1014
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(1142..1158)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1142..1158)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1155..1177)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1166..1182
                     /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(1190..1196,1197..1214,1215..1220))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1235..1256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1373..1390)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1544..2132)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1624..1643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2310..3119)
                     /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     3033..3052
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3111..3130
                     /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 ggagagagcc gccaccatgg gtaagcctat ccctaaccct
      481 ctcctcggtc tcgattctac gatggtcttt acactcgaag atttcgttgg ggactggcga
      541 cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt
      601 cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg tgaaaatggg
      661 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga ccaaatgggc
      721 cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc
      781 ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga ctatttcgga
      841 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg
      901 tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc
      961 cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct ggcgacgcgt
     1021 gctgaagctg ctgcaaagga agctgcagct aaggaggctg cagctaaggc tggcagcagt
     1081 cttcatcgga tcccgggccc gtcgactgca gaggcctgca tgcaagcttg gtgtaatcat
     1141 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     1201 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1261 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1321 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1381 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1441 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1501 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1561 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1621 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1681 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1741 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1801 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1861 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1921 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1981 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2041 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2101 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2161 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2221 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaagc ccaatctgaa
     2281 taatgttaca accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac
     2341 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     2401 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     2461 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     2521 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     2581 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     2641 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     2701 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     2761 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgtttttccg
     2821 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     2881 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     2941 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaag
     3001 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3061 tcagcatcca tgttggaatt taatcgcggc ctcgacgttt cccgttgaat atggctcata
     3121 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     3181 ttatcttgtg caatgtaaca tcagagattt tgagacacgg gccagagctg ca
//
