LOCUS       sequence-431510-        7156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (C' terminus) for prime editing; using PEmax; for 
            PE4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7156)
  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 7156)
  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: AA185
FEATURES             Location/Qualifiers
     source          1..7156
                     /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"
     CDS             495..515
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2616..2636
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2646..2672
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             2682..2738
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(5066..5082)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5066..5082)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5079..5101)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5090..5106
                     /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(5114..5120,5121..5138,5139..5144))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5159..5180
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5297..5314)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5468..6056)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5548..5567)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6234..7043)
                     /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     6957..6976
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7035..7054
                     /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 tgaagacggg tccggaagct ctggtggcag caagcggacc gccgacggct
      481 ctgaattcga gagccctaag aagaaaagaa aggtgagcgg aggctctagc ggcggaagca
      541 ccctgaacat tgaggacgag tatagactgc atgaaacaag caaggaaccc gacgtgtccc
      601 tgggctccac ctggctgtcc gactttcccc aggcctgggc cgagacagga ggaatgggcc
      661 tggccgtgcg gcaggcaccc ctgatcatcc ctctgaaggc cacctctaca cccgtgagca
      721 tcaagcagta ccctatgtct caggaggcca gactgggcat caagcctcac atccagaggc
      781 tgctggacca gggcatcctg gtgccatgcc agagcccctg gaacacacca ctgctgcccg
      841 tgaagaagcc aggcaccaat gactatagac ccgtgcagga tctgagagag gtgaacaaga
      901 gggtggagga tatccacccc accgtgccca acccttacaa tctgctgtcc ggcctgcccc
      961 cttctcacca gtggtataca gtgctggacc tgaaggatgc cttcttttgt ctgagactgc
     1021 accctaccag ccagccactg ttcgcctttg agtggaggga ccctgagatg ggcatctctg
     1081 gccagctgac ctggacacgc ctgcctcagg gcttcaagaa tagcccaaca ctgtttaacg
     1141 aggccctgca ccgcgacctg gcagatttcc ggattcagca cccagatctg atcctgctgc
     1201 agtacgtgga cgatctgctg ctggccgcca ccagcgagct ggattgccag cagggaacac
     1261 gcgccctgct gcagaccctg ggaaacctgg gatatagggc atccgccaag aaggcccaga
     1321 tctgtcagaa gcaggtgaag tacctgggct atctgctgaa ggagggccag agatggctga
     1381 cagaggccag gaaggagaca gtgatgggcc agccaacacc caagacccca agacagctga
     1441 gggagttcct gggcaaagca ggattttgca ggctgttcat cccaggattc gcagagatgg
     1501 cagcacctct gtacccactg accaagccgg gcaccctgtt taattggggc cctgaccagc
     1561 agaaggccta tcaggagatc aagcaggccc tgctgacagc accagccctg ggcctgccag
     1621 acctgaccaa gcctttcgag ctgtttgtgg atgagaagca gggctacgcc aagggcgtgc
     1681 tgacccagaa gctgggacca tggaggcggc ccgtggccta tctgtccaag aagctggacc
     1741 cagtggcagc aggatggcca ccatgcctga ggatggtggc agcaatcgcc gtgctgacaa
     1801 aggatgccgg caagctgacc atgggacagc cactggtcat cctggcacca cacgcagtgg
     1861 aggccctggt gaagcagcct ccagatcgct ggctgtctaa cgcccggatg acacactacc
     1921 aggccctgct gctggacacc gatcgcgtgc agtttggccc tgtggtggcc ctgaatccag
     1981 ccaccctgct gcctctgcca gaggagggcc tgcagcacaa ctgtctggac atcctggcag
     2041 aggcacacgg aacaaggcca gacctgaccg atcagcccct gcctgacgcc gatcacacat
     2101 ggtataccga tggaagctcc ctgctgcagg agggccagag gaaggcagga gcagcagtga
     2161 ccacagagac agaagtgatc tgggccaagg ccctgccagc aggcacatcc gcccagcggg
     2221 ccgagctgat cgccctgacc caggccctga agatggccga gggcaagaag ctgaacgtgt
     2281 acacagactc cagatatgcc ttcgccaccg cacacatcca cggagagatc tacaggcgcc
     2341 ggggctggct gacctctgag ggcaaggaga tcaagaacaa ggatgagatc ctggccctgc
     2401 tgaaggccct gtttctgccc aagcggctga gcatcatcca ctgtcctgga caccagaagg
     2461 gacactccgc cgaggcaagg ggcaatcgga tggccgacca ggccgccaga aaggctgcta
     2521 ttactgaaac tcccgacact tccactctgc tgattgaaaa ctcctcccct tctggcggct
     2581 caaaaagaac cgccgacggc agcgaattcg agtctcccaa gaagaagagg aaagtcggat
     2641 ccggccctgc cgctaagaga gtgaagctgg acggaagcgg agctactaac ttcagcctgc
     2701 tgaagcaggc tggggacgtg gaggagaacc ctggacctag cttcgttgct ggagtcatcc
     2761 ggagactgga cgagacagtg gtgaacagaa ttgccgccgg cgaggtgatc cagagacctg
     2821 ccaatgcaat aaaggagatg atcgagaact gtctggacgc caagtccaca agcattcagg
     2881 tgatcgtgaa ggagggcgga ctgaagctga tccagatcca agacaacggc acaggcatca
     2941 gaaaggaaga tctggacatc gtgtgtgaac ggttcaccac atctaagctg cagtcttttg
     3001 aggatctggc ctctatcagt acctacggct tcagaggcga ggccctggcc agcatcagcc
     3061 acgtggccca tgtgaccatc accaccaaaa ccgccgacgg caaatgcgct tatcgcgcta
     3121 gctacagcga cggcaagctg aaagccccgc caaagccttg cgccggcaac cagggtacac
     3181 agataacagt ggaggatctg ttctacaaca tcgccacccg gagaaaggcc ctgaaaaatc
     3241 ccagcgagga gtacggcaag atcctggaag tcgtcggcag atactccgtg cacaacgccg
     3301 gaatcagctt tagcgtaaag aagcagggag aaaccgtggc cgatgtgcgc accctgccaa
     3361 atgccagcac cgtggataac atcagaagca ttttcggaaa tgccgtgtcc agagaactga
     3421 tcgagatcgg ctgcgaagat aagaccctgg cttttaagat gaacggctac atcagcaacg
     3481 ccaattactc tgtgaagaag tgcatctttc ttctgttcat caaccacaga ctggtggaaa
     3541 gcaccagcct gcggaaagcc atcgagacag tgtacgccgc ctacctgcct aagaacaccc
     3601 accccttcct gtacctgagc ctcgagatca gccctcagaa cgtggacgtc aatgtgcatc
     3661 ctacaaagca cgaggtgcac ttcctgcacg aggaaagcat cctggaaaga gtgcagcagc
     3721 acattgagag caagctgctg ggctctaaca gcagcagaat gtacttcaca cagaccctgc
     3781 tgcctggcct ggccggcccc tcaggcgaaa tggttaagtc cacaacctct ctgacctcat
     3841 ccagcaccag cggttcttcc gataaggtgt acgcccacca gatggtgcgg accgactctc
     3901 gggagcagaa gctggacgcc tttctgcaac ctctgagcaa acctctgagc tctcagcctc
     3961 aggccatcgt gaccgaggac aagacagata tctcctccgg ccgtgccaga cagcaggacg
     4021 aagaaatgct cgagctgcca gctcctgccg aggtggccgc caagaaccag agcctggagg
     4081 gagataccac aaagggcacc agcgaaatga gcgagaagcg gggccctacc tccagcaacc
     4141 ccagaaaacg gcaccgggag gacagcgacg tggaaatggt ggaggacgac agcagaaagg
     4201 aaatgacagc cgcttgtacc cctagaagaa gaatcatcaa cctgacctcc gtgctgagcc
     4261 tgcaggagga gatcaacgag cagggccacg aggtgctgag agagatgctg cacaatcaca
     4321 gcttcgtggg ctgcgtgaac cctcaatggg ccctggctca gcatcaaaca aagctgtacc
     4381 tgctgaacac caccaagctg agcgaagagc tgttctacca gatcctcatc tacgacttcg
     4441 ccaacttcgg cgtgctacgc ctgagcgagc ccgcccctct gtttgacctg gccatgctgg
     4501 ctctggatag cccagaaagc ggctggacag aagaggacgg acctaaagag gggctggctg
     4561 aatacatcgt ggagttcctg aagaaaaagg ccgagatgct ggccgactac ttttctctgg
     4621 aaatcgacga ggaaggcaac ctgatcggcc tgcctctgct gatcgataac tacgtgcctc
     4681 ccctggaagg cctgcccatc ttcatcctga gactggctac agaggtgaac tgggacgagg
     4741 aaaaggaatg cttcgagtct ctgagcaagg agtgcgccat gttctatagc atcagaaaac
     4801 agtacatctc tgaagagagc actctgtctg gccagcagag tgaagtgccc ggaagcatcc
     4861 ccaacagctg gaagtggacc gtggaacaca tcgtgtacaa ggccctgcgg agccacattc
     4921 tccctcctaa gcacttcacc gaggacggca acatcctgca gctggccaac ctgcccgacc
     4981 tttataaggt tttcggaagc acgtcttcat cggatcccgg gcccgtcgac tgcagaggcc
     5041 tgcatgcaag cttggtgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5101 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5161 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5221 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5281 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5341 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5401 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5461 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5521 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5581 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5641 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5701 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5761 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5821 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5881 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5941 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6001 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6061 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6121 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6181 gttttaaatc aagcccaatc tgaataatgt tacaaccaat taaccaattc tgattagaaa
     6241 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     6301 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     6361 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     6421 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     6481 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     6541 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     6601 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     6661 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     6721 aatacctgga atgctgtttt tccggggatc gcagtggtga gtaaccatgc atcatcagga
     6781 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     6841 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     6901 ggcgcatcgg gcttcccata caagcgatag attgtcgcac ctgattgccc gacattatcg
     6961 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgac
     7021 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     7081 tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac
     7141 acgggccaga gctgca
//
