LOCUS       sequence-431481-        3125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) guide expression; reverse 
            orientation; positive control cell surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3125)
  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 3125)
  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: AA324
FEATURES             Location/Qualifiers
     source          1..3125
                     /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"
     promoter        complement(648..896)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(706..725)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(876..896)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1035..1051)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1035..1051)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1048..1070)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1059..1075
                     /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(1083..1089,1090..1107,1108..1113))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1128..1149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1266..1283)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1437..2025)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1517..1536)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2203..3012)
                     /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     2926..2945
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3004..3023
                     /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 tgaagacgtc agaaccgact cggtgccact ttttcaaaaa aaaccttacc
      481 gtccccagca ggcactatct actagagtag aaatttagtg ccgtccaggt tacagaagaa
      541 tctacgatag tagaaattag tagctgagct gatcctggaa agatctacga cagtagaaat
      601 tagtctgtga cagtaacagt acttgatcta caagagtaga aattaccggt gtttcgtcct
      661 ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga
      721 tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta
      781 cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattct aattatctct
      841 ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcttcg
      901 aatctagttg caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac
      961 cttctacacg cgtcttcatc ggatcccggg cccgtcgact gcagaggcct gcatgcaagc
     1021 ttggtgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1081 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1141 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1201 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1261 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1321 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1381 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1441 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1501 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1561 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1621 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1681 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1741 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1801 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1861 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1921 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1981 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2041 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2101 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2161 agcccaatct gaataatgtt acaaccaatt aaccaattct gattagaaaa actcatcgag
     2221 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     2281 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     2341 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     2401 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     2461 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     2521 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     2581 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     2641 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     2701 tgctgttttt ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     2761 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     2821 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     2881 cttcccatac aagcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     2941 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgacg tttcccgttg
     3001 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     3061 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca cgggccagag
     3121 ctgca
//
