LOCUS       sequence-432906-        5682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Unfused click editor construct expressing mSA-ePhi29(D169A), 
            expressed from CMV or T7 promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5682)
  AUTHORS   Ferreira da Silva J, Tou CJ, King EM, Eller ML, Rufino-Ramos D, Ma 
            L, Cromwell CR, Metovic J, Benning FMC, Chao LH, Eichler FS, 
            Kleinstiver BP
  TITLE     Click editing enables programmable genome writing using DNA 
            polymerases and HUH endonucleases.
  JOURNAL   Nat Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x.
  PUBMED    39039307
REFERENCE   2  (bases 1 to 5682)
  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: "Nat 
            Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x."
COMMENT     Sequence Label: Unfused click editor construct; mSA-ePhi29 -
            p...
FEATURES             Location/Qualifiers
     source          1..5682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1215..1234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1406..1785
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1786..1989
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1940..1960
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2031..2050
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2031..2049
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2057..2066
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2090..2431
                     /codon_start=1
                     /product="monomeric streptavidin/rhizavidin hybrid (Lim et 
                     al., 2013)"
                     /label=mSA
                     /translation="AEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRAQGTGCQNSPY
                     TLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATE
                     QGQDTFTKVK"
     CDS             2507..2527
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4322..4342
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4351..4368
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4391..4408)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4397..4621
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4692..4708)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4692..4708)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4705..4727)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4716..4732
                     /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(4740..4746,4747..4764,4765..4770))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4785..4806
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4923..4940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5094..5682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5174..5193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gcgaaccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta
     1201 gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     1261 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     1321 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     1381 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     1441 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1501 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1561 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1621 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1681 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1741 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     1801 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1861 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1921 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1981 agcagagctg gtttagtgaa ccgtcagatc cgctagagat ccgcggccgc taatacgact
     2041 cactataggg agagccgcca ccatgggggc ccagccggcc agatctatgg cggaagcggg
     2101 tatcaccggc acgtggtaca accagtctgg ttctaccttc accgttaccg cgggtgcgga
     2161 cggtaacctg accggtcagt acgaaaaccg tgcgcagggc actggttgcc agaactctcc
     2221 gtacaccctg accggtcgtt acaacggtac caaactggaa tggcgtgttg aatggaacaa
     2281 ctctaccgaa aactgccact ctcgtaccga atggcgtggt cagtaccagg gtggtgcgga
     2341 agcgcgtatc aacacccagt ggaacctgac ctacgaaggt ggttctggtc cggcgaccga
     2401 acagggtcag gacaccttca ccaaagttaa accgtctgcg gcgtctggat ccagcgctgg
     2461 aggcggtggc tccggaggag gcggaagcgg aggaggaggt agcggaccta agaaaaagag
     2521 gaaggtggcg gccgctggat ccaagcacat gcctagaaag agatacagct gcgacttcga
     2581 gacaacaacc aaggtcgagg actgcagagt gtgggcttac ggatatatga atatcgagga
     2641 ccactctgag tacaagatcg gaaatagcct ggatgagttt atggcctggg ccctgaaagt
     2701 gcaggccgac ctgtacttcc acaacctgaa attcgacggc gccttcatca ttaactggct
     2761 ggaacggaac ggctttaagt ggtccgccga tggactgcca aacacctaca acaccatcat
     2821 cagcagaacc ggccagtggt acatgatcga catctgcctg ggctacaagg gcaaaagaaa
     2881 gatccatacc gtgatctacg acagcctgaa gaaactgcct ttccccgtga agaagatcgc
     2941 taaagacttc aagctgacag tgctgaaggg cgacattgat taccacaagg aaagacctgt
     3001 gggatacaag atcacacctg aggaatatgc ctacatcaag aacgccatcc agatcatcgc
     3061 cgaggccctg ctgatccagt tcaagcaggg cctggaccgg atgaccgctg gcagcgacag
     3121 cctcaaagac ttcaaggaca ttatcaccac taagaagttc aaaaaggtgt tcccaacact
     3181 gtctcttgga ctggacaaga aggtgcggta cgcctataga ggcggcttta cctggctgaa
     3241 cgacagattc aaggaaaagg aaatcggcga gggcatggtg tttgacgtga actccctcta
     3301 ccctgcccag atgtacagcc ggctgctgcc ctatggcgag cctatcgtgt ttgagggcaa
     3361 gtacgtgtgg gatgaagatt accccctgca catccagcac atccggtgcg agttcgagct
     3421 gaaggagggc tacatcccaa caatccaaat caaacggagc agattctaca agggcaacga
     3481 gtacctgaaa agcagcggcg gcgaaatcgc cgatctgtgg ctgagcaatg tggacctgga
     3541 actgatgaag gaacactacg acctgtacaa cgtggaatac atcagcggcc tgaagtttaa
     3601 ggccacaacc ggcctgttca aagacttcat cgataagtgg acctacatca aaacaaccag
     3661 cgagggcgct atcaagcagc tggccaagct gatgctgaat tctctgtacg gcaagttcgc
     3721 cagcaaccct gatgtcacag gcaaggtgcc ctacctgaag gaaaacggcg ccctgggctt
     3781 cagactgggc gaggaagaaa ccaaggaccc tgtgtacacc cctatgggtg ttttcatcac
     3841 cgcctgggcc agatacacca ccatcaccgc cgctcaggct tgttacgata gaatcatcta
     3901 ttgtgatacc gactctatcc acctgaccgg gaccgagatc cccgacgtga tcaaggatat
     3961 tgtggacccc aagaaactgg gctactgggc ccacgagtcc accttcaagc gggccaagta
     4021 cctgagaccc aagacctaca tccaggacat ctacatgaaa gaagtggacg gcgagctcgt
     4081 ggaaggcagc cccgacgact acacagacat caagctgagc gtgaagtgcg ccggcatgac
     4141 cgataagatc aaaaaggagg tgacatttga gaacttcaag gtgggattca gccgcaagat
     4201 gaaacctaag cctgttcaag tgcctggcgg tgttgtgctg gtggatgaca ccttcacaat
     4261 caagtccggc ggaagctctg gtggcagcaa gcggaccgcc gacggctctg aattcgagag
     4321 ccctaagaag aaaagaaagg tgtgaccggt catcatcacc atcaccattg agtttaaacc
     4381 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4441 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4501 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4561 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     4621 gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg
     4681 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4741 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc
     4801 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4861 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4921 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4981 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5041 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5101 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5161 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5221 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5281 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5341 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5401 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5461 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5521 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5581 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5641 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aa
//
