LOCUS       sequence-343630-        6303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human MLH1dn (codon optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6303)
  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 6303)
  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-hMLH1dn
FEATURES             Location/Qualifiers
     source          1..6303
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        193..1371
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          424..1362
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1319..1339
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3663..3680
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3703..3720)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3709..3933
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4004..4020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4004..4020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4017..4039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4028..4044
                     /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(4052..4058,4059..4076,4077..4082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4097..4118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4235..4252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4406..4994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4486..4505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5165..5956,5957..6025))
                     /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     5788..5807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6026..6130)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6209..6228)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
       61 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actgcaaaga
      121 tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa
      181 ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
      241 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
      301 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
      361 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
      421 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
      481 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
      541 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
      601 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
      661 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
      721 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
      781 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
      841 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
      901 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
      961 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
     1021 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     1081 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     1141 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg
     1201 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     1261 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     1321 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg acgtacggga
     1381 tcctctagag ccaccatgag cttcgttgct ggagtcatcc ggagactgga cgagacagtg
     1441 gtgaacagaa ttgccgccgg cgaggtgatc cagagacctg ccaatgcaat aaaggagatg
     1501 atcgagaact gtctggacgc caagtccaca agcattcagg tgatcgtgaa ggagggcgga
     1561 ctgaagctga tccagatcca agacaacggc acaggcatca gaaaggaaga tctggacatc
     1621 gtgtgtgaac ggttcaccac atctaagctg cagtcttttg aggatctggc ctctatcagt
     1681 acctacggct tcagaggcga ggccctggcc agcatcagcc acgtggccca tgtgaccatc
     1741 accaccaaaa ccgccgacgg caaatgcgct tatcgcgcta gctacagcga cggcaagctg
     1801 aaagccccgc caaagccttg cgccggcaac cagggtacac agataacagt ggaggatctg
     1861 ttctacaaca tcgccacccg gagaaaggcc ctgaaaaatc ccagcgagga gtacggcaag
     1921 atcctggaag tcgtcggcag atactccgtg cacaacgccg gaatcagctt tagcgtaaag
     1981 aagcagggag aaaccgtggc cgatgtgcgc accctgccaa atgccagcac cgtggataac
     2041 atcagaagca ttttcggaaa tgccgtgtcc agagaactga tcgagatcgg ctgcgaagat
     2101 aagaccctgg cttttaagat gaacggctac atcagcaacg ccaattactc tgtgaagaag
     2161 tgcatctttc ttctgttcat caaccacaga ctggtggaaa gcaccagcct gcggaaagcc
     2221 atcgagacag tgtacgccgc ctacctgcct aagaacaccc accccttcct gtacctgagc
     2281 ctcgagatca gccctcagaa cgtggacgtc aatgtgcatc ctacaaagca cgaggtgcac
     2341 ttcctgcacg aggaaagcat cctggaaaga gtgcagcagc acattgagag caagctgctg
     2401 ggctctaaca gcagcagaat gtacttcaca cagaccctgc tgcctggcct ggccggcccc
     2461 tcaggcgaaa tggttaagtc cacaacctct ctgacctcat ccagcaccag cggttcttcc
     2521 gataaggtgt acgcccacca gatggtgcgg accgactctc gggagcagaa gctggacgcc
     2581 tttctgcaac ctctgagcaa acctctgagc tctcagcctc aggccatcgt gaccgaggac
     2641 aagacagata tctcctccgg ccgtgccaga cagcaggacg aagaaatgct cgagctgcca
     2701 gctcctgccg aggtggccgc caagaaccag agcctggagg gagataccac aaagggcacc
     2761 agcgaaatga gcgagaagcg gggccctacc tccagcaacc ccagaaaacg gcaccgggag
     2821 gacagcgacg tggaaatggt ggaggacgac agcagaaagg aaatgacagc cgcttgtacc
     2881 cctagaagaa gaatcatcaa cctgacctcc gtgctgagcc tgcaggagga gatcaacgag
     2941 cagggccacg aggtgctgag agagatgctg cacaatcaca gcttcgtggg ctgcgtgaac
     3001 cctcaatggg ccctggctca gcatcaaaca aagctgtacc tgctgaacac caccaagctg
     3061 agcgaagagc tgttctacca gatcctcatc tacgacttcg ccaacttcgg cgtgctacgc
     3121 ctgagcgagc ccgcccctct gtttgacctg gccatgctgg ctctggatag cccagaaagc
     3181 ggctggacag aagaggacgg acctaaagag gggctggctg aatacatcgt ggagttcctg
     3241 aagaaaaagg ccgagatgct ggccgactac ttttctctgg aaatcgacga ggaaggcaac
     3301 ctgatcggcc tgcctctgct gatcgataac tacgtgcctc ccctggaagg cctgcccatc
     3361 ttcatcctga gactggctac agaggtgaac tgggacgagg aaaaggaatg cttcgagtct
     3421 ctgagcaagg agtgcgccat gttctatagc atcagaaaac agtacatctc tgaagagagc
     3481 actctgtctg gccagcagag tgaagtgccc ggaagcatcc ccaacagctg gaagtggacc
     3541 gtggaacaca tcgtgtacaa ggccctgcgg agccacattc tccctcctaa gcacttcacc
     3601 gaggacggca acatcctgca gctggccaac ctgcccgacc tttataaggt tttctaaccg
     3661 gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta
     3721 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3781 ctcccactgt cctttcctaa taaaatgaga aaattgcatc gcattgtctg agtaggtgtc
     3841 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3901 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3961 gctcgatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4021 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4081 aagcctaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4141 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4201 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4261 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4321 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4381 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4441 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4501 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4561 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4621 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4681 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4741 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4801 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4861 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4921 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4981 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacact cagtggaacg
     5041 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5101 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5161 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5221 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5281 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5341 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5401 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5461 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5521 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5581 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5641 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5701 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5761 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5821 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5881 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5941 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6001 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6061 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6121 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatcgat
     6181 ctcccgatcc cctagggtcg actctcagta caatctgctc tgatgccgca tagttaagcc
     6241 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     6301 cta
//
