LOCUS       sequence-343633-        5109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human MLH1NTD-NLS (codon optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5109)
  AUTHORS   Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, Chen
            C, Nelson JW, Newby GA, Sahin M, Osborn MJ, Weissman JS, Adamson B, 
            Liu DR
  TITLE     Enhanced prime editing systems by manipulating cellular determinants
            of editing outcomes.
  JOURNAL   Cell. 2021 Oct 28;184(22):5635-5652.e29. doi: 
            10.1016/j.cell.2021.09.018.
  PUBMED    34653350
REFERENCE   2  (bases 1 to 5109)
  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"; doi: 
            "10.1016/j.cell.2021.09"; journalName: "Cell"; date: "2021-10-28- 
            28"; volume: "184"; issue: "22"; pages: "5635-5652.e29"
COMMENT     Sequence Label: pEF1a-hMLH1NTD-NLS
FEATURES             Location/Qualifiers
     source          1..5109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        134..1312
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          365..1303
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1260..1280
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2381..2401
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2410..2427
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2450..2467)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2456..2680
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2751..2767)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2751..2767)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2764..2786)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2775..2791
                     /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(2799..2805,2806..2823,2824..2829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2844..2865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2982..2999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3153..3741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3233..3252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3912..4703,4704..4772))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4535..4554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4773..4877)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4956..4975)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactgcaaag
       61 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
      121 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
      181 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
      241 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
      301 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
      361 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
      421 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt
      481 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg
      541 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
      601 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
      661 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc
      721 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
      781 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc
      841 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
      901 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
      961 ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     1021 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     1081 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg
     1141 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     1201 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     1261 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gacgtacggg
     1321 atcctctaga gccaccatga gcttcgttgc tggagtcatc cggagactgg acgagacagt
     1381 ggtgaacaga attgccgccg gcgaggtgat ccagagacct gccaatgcaa taaaggagat
     1441 gatcgagaac tgtctggacg ccaagtccac aagcattcag gtgatcgtga aggagggcgg
     1501 actgaagctg atccagatcc aagacaacgg cacaggcatc agaaaggaag atctggacat
     1561 cgtgtgtgaa cggttcacca catctaagct gcagtctttt gaggatctgg cctctatcag
     1621 tacctacggc ttcagaggcg aggccctggc cagcatcagc cacgtggccc atgtgaccat
     1681 caccaccaaa accgccgacg gcaaatgcgc ttatcgcgct agctacagcg acggcaagct
     1741 gaaagccccg ccaaagcctt gcgccggcaa ccagggtaca cagataacag tggaggatct
     1801 gttctacaac atcgccaccc ggagaaaggc cctgaaaaat cccagcgagg agtacggcaa
     1861 gatcctggaa gtcgtcggca gatactccgt gcacaacgcc ggaatcagct ttagcgtaaa
     1921 gaagcaggga gaaaccgtgg ccgatgtgcg caccctgcca aatgccagca ccgtggataa
     1981 catcagaagc attttcggaa atgccgtgtc cagagaactg atcgagatcg gctgcgaaga
     2041 taagaccctg gcttttaaga tgaacggcta catcagcaac gccaattact ctgtgaagaa
     2101 gtgcatcttt cttctgttca tcaaccacag actggtggaa agcaccagcc tgcggaaagc
     2161 catcgagaca gtgtacgccg cctacctgcc taagaacacc caccccttcc tgtacctgag
     2221 cctcgagatc agccctcaga acgtggacgt caatgtgcat cctacaaagc acgaggtgca
     2281 cttcctgcac gaggaaagca tcctggaaag agtgcagcag cacattgaga gcaagctgct
     2341 gggctctaaa cggacagccg acggaagcga gttcgagtca ccaaagaaga agcggaaagt
     2401 ctaaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
     2461 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2521 ggtgccactc ccactgtcct ttcctaataa aatgagaaaa ttgcatcgca ttgtctgagt
     2581 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2641 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2701 agctggggct cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     2761 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2821 aagtgtaaag cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2881 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2941 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3001 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3061 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3121 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3181 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3241 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3301 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3361 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3421 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3481 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3541 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3601 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3661 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3721 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacactcag
     3781 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3841 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3901 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3961 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4021 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4081 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4141 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4201 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4261 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4321 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4381 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4441 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4501 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4561 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4621 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4681 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4741 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4801 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4861 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     4921 gatcgatctc ccgatcccct agggtcgact ctcagtacaa tctgctctga tgccgcatag
     4981 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     5041 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     5101 aggcgtttt
//
