LOCUS       sequence-431519-        3607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (N' terminus) conditional degradation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3607)
  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 3607)
  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: AA414
FEATURES             Location/Qualifiers
     source          1..3607
                     /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..1521)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHAGLCSRRARDPMKTAASLSCSLLSCSFLCSSFSTRA
                     GNLERRQPARDLAADQVRVIFLQQIQTRADVHSLAVRQAFGQASGFGRWDQNAWGAPEQ
                     QLRDRGLVQPVHVFFDQRDDVRWLTGDWQLVWEAPQWLAVNRIQERLAVMVHLDFSQRA
                     DDTHRQRTLDKNVLIDDQLAADVGGAEGLEGLAGKFWPFVPGRDRADELHKCNTFDALW
                     IALGPVETQSGAPVVNDQDDLFQTQGFDEGIHEADVVVEEITQVWFVGFAHTDQVWSNA
                     AMGRCNMRDDVAPHVGGGRVTVQEQHRGAIARTNIDVVHALAQDFHIMGVEWKASTDFC
                     RRIETEERVRDRLTHGGGSLLRLHLDVFARYRARILWPSFYNVVTGKTLALPNLIALQH
                     IPLSPAGVIAKRPAPIALPNSCAA"
     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..1389
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     primer_bind     complement(1517..1533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1517..1533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1530..1552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1541..1557
                     /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(1565..1571,1572..1589,1590..1595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1610..1631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1748..1765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1919..2507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1999..2018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2685..3494)
                     /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     3408..3427
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3486..3505
                     /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 ggcagaaatc ggtactggct ttccattcga cccccattat
      541 gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct
      601 gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat
      661 gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa
      721 ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc
      781 ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac
      841 tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct
      901 atcccgacct gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc
      961 accgacgtcg gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg
     1021 atgggtgtcg tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg
     1081 aatcctgttg accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag
     1141 ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc
     1201 ccgaagctgc tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc
     1261 ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg
     1321 caagaagata acccggacct gatcggcagc gagatcgcgc gctggctgtc gacgctcgag
     1381 atttccggca cgcgtgctga agctgctgca aaggaagctg cagctaagga ggctgcagct
     1441 aaggctggca gcagtcttca tcggatcccg ggcccgtcga ctgcagaggc ctgcatgcaa
     1501 gcttggtgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1561 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1621 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1681 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1741 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1801 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1861 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1921 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1981 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2041 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2101 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2161 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2221 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2281 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2341 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2401 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2461 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2521 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2581 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2641 caagcccaat ctgaataatg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg
     2701 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2761 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2821 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     2881 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     2941 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     3001 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     3061 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     3121 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     3181 aatgctgttt ttccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     3241 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     3301 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     3361 ggcttcccat acaagcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     3421 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga cgtttcccgt
     3481 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     3541 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacgggccag
     3601 agctgca
//
