LOCUS       sequence-213346-        5837 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for knock-in HaloTag fusing to the N-terminal of mouse
            Erk2 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5837)
  AUTHORS   Gu B, Posfai E, Rossant J
  TITLE     Efficient generation of targeted large insertions by microinjection 
            into two-cell-stage mouse embryos.
  JOURNAL   Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166.
  PUBMED    29889212
REFERENCE   2  (bases 1 to 5837)
  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. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166."
COMMENT     Sequence Label: Halo-Erk2 donor
FEATURES             Location/Qualifiers
     source          1..5837
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(16..35)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     226..247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        410..514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(515..583,584..1375)
                     /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     complement(733..752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1546..2134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2035..2054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2288..2305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2422..2443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2458..2463,2464..2481,2482..2488)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2496..2512
                     /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)."
     primer_bind     2501..2523
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2520..2536
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2520..2536
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2555..2575
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2557..2575
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2604..2620
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2605..2621
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3499..3519
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             3535..4419
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(5578..5597)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5579..5597)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5607..5624)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5607..5623)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5616..5638)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
       61 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      121 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      181 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
      241 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
      301 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
      361 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      481 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
      541 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
      601 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
      661 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
      721 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
      781 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
      841 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
      901 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
      961 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1021 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     1081 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1141 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1201 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     1261 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     1321 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     1381 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     1441 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     1501 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1561 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     1621 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     1681 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     1741 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     1801 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     1861 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     1921 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     1981 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2041 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2101 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2161 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2221 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2281 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     2341 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2401 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     2461 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     2521 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     2581 aaagctgggt accgggcccc ccctcgaggt cgacggtatc gataagcttg atatcgaatt
     2641 ctctgcttat attctgtcca gtgggcatca tggggttcta ggactgtatc tggttatctt
     2701 gcgtgagtga tgtcactgag ctctggatca ggagcagcct tggatgctgt gggattcctg
     2761 cttaatctag ttttatatgt ctaaaaggca ttttttaaaa ttatgctcat tacagggtga
     2821 aagttttttt ctgggtctgg gttatgcttt ttggtttctg tcccactctg atggtctaag
     2881 ttcatcagag tgcacaatgt gggccacgta ttcttagagg tctgtctcag gggtcccgtg
     2941 gttggtgtgc agtgagtcct ctggtctcaa gccaagtccc tggtcccgtg tgagttcgtt
     3001 ctggaccagg cacagcacca ggacagtggg tgggactctg tggggtcctt atgcctaaat
     3061 tccaacctag ctctctagag ctacccagcg cttgggtctc cgagcgcaca tgacgtccct
     3121 cccccgcagc cgcagtgctc ctgcctcacc tcgctgcttc cgccgacccg cgagactccg
     3181 ccctctctac caagagaagc caatcaacac gcacgccctt gactccgccc tagcgccctt
     3241 cgacgcgaac ccttccctcc tcccactcgt agcccgcccg tcaggcagga aggctggcag
     3301 tggttctacc ggcggttaat tctctcctct gtgttgtcct ccttcctcgt tcccgatcgc
     3361 cggcgggggc ggctacacgg gcggcagcgc ggttcctgcg ggaagcgcag cataagtcga
     3421 gcggcagccg cgaagcgtcg aaccgaacgc ggcggcggcg gcggcggcgg cggctgtgca
     3481 gccaacatgg aattcatgga ggatctgtac tttcagagcg ataacgatgg atccgaaatc
     3541 ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac
     3601 gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc
     3661 tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca
     3721 gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt cgacgaccac
     3781 gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt
     3841 cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa
     3901 ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt
     3961 gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat
     4021 cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct gactgaagtc
     4081 gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc actgtggcgc
     4141 ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa
     4201 tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg caccccaggc
     4261 gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct
     4321 gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct gatcggcagc
     4381 gagatcgcgc gctggctgtc tactctggag atttccggtg gtagcggcag cggtagcact
     4441 agttgtgcag ccaacatggc ggcggcggcg gcggcgggcc cggagatggt ccgcgggcag
     4501 gtgttcgacg tagggccgcg ctacaccaac ctctcgtaca tcggagaagg cgcctacggc
     4561 atggtttggt gagtatccgc gctggatttc aggcccggcc tcacgcggtc accctgtgcc
     4621 ccctgcctgt ggctgatctg gacgcaccca cggcccttcc cgcgccttgg cctcgcgttc
     4681 tgcggcgccc cggacccctg gcttcttggc cttcactcct ggagccctgg ggcggggtcc
     4741 tggagccggt cgaggcttag gtgttgctcc tcgtcttcct ctgctgcttc tcgtttgcgg
     4801 ttggaggggc aaccccgatt gaggccagga ctcgaagact ccggggcttg gtcttaaagc
     4861 gcgtactttc gaggcttctc cctcgggacg ggaacggaag tacactgtgt ctcctaattt
     4921 atagcattat agccaccgcc tcttgacatt agattaaagc caggaattgt attagatgct
     4981 tgtccgtggt ggcggcggcc atggttttgc aagacggaat aataggcttg tgtgctgagc
     5041 accaaaatat ttttcttctc ttctgtgtac gtgaacagca ttcacaaaat atcttaagta
     5101 gcgctaatga tgctttctgc ctcgggctta accgttaatg acacagcgtt ggttgtggac
     5161 atattgaaat cttgaagttt caaaatggat ttaaatatac cgattgtgtt tcagaaagga
     5221 taagccagca tcactctaga attattaaga agtatttgga gttagtccta atttgacttt
     5281 aaattatccc gtcgttaccc tcaagagtac tctcttaaca gtgtgaatgt atccatatgt
     5341 tttagcccga ttactgtgat cgtttcccca taacgtttca gtgttcaaga atagtttttg
     5401 cacgatcact tgcaggattt cctgcatgtg gaatgttgaa gtgtattgtt ttaactaaaa
     5461 caagttcatt tatcgaaggt ctctgagata gtttaattta acttagctat cttgaggaaa
     5521 gagaagacag ccttactagt tctagagcgg ccgccaccgc ggtggagctc caattcgccc
     5581 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     5641 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     5701 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     5761 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5821 accgctacac ttgccag
//
