LOCUS       sequence-217907-        5836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for knock-in HaloTag fusing to the N-terminal of mouse
            Erk2 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5836)
  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 5836)
  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..5836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(898..918)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(1796..1812)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1797..1813)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1842..1862)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1842..1860)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1881..1897)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1881..1897)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1894..1916)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1905..1921
                     /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(1929..1935,1936..1953,1954..1959))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1974..1995
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2112..2129)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2283..2871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2363..2382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3042..3833,3834..3902))
                     /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     3665..3684
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3903..4007)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4034..4489
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4121..4140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4331..4352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4615..4637
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4629..4646
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4630..4646
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4656..4675
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4656..4674
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(5834..5836,1..882))
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
ORIGIN
        1 ggaaatctcc agagtagaca gccagcgcgc gatctcgctg ccgatcaggt ccgggttgtc
       61 ttcttgcagc agattcagac ccgggccgat gtccacagcc ttgcagttag gcaggctttt
      121 ggccaggcga gcggcttcgg ccggtgggat cagaacgcct ggggtgcccc agaacagcag
      181 cttcgggaca ggggactggt gcagccagtc catgtattct tcgaccagcg cgacgatgtt
      241 cgctggctca ccggcgattg gcagctcgtt tgggaagcgc cacagtggct cgcggtcaac
      301 aggattcagg aacggctcgc ggtaatggtc catctcgact tcagtcagcg ggcggacgac
      361 acccatcggc agcgtaccct cgataaaaac gttctgatcg atgatcagct tgcggccgac
      421 gtcggtggtg cggaaggcct ggaaggtctc gcgggcaaat tctggccatt cgtcccaggt
      481 cgggataggg cggatgaact ccataaatgc aatacctttg acgcgctctg gattgcgctt
      541 ggcccagtgg aaacccagag cggagcccca gtcgtgaatg accaggacga cctcttccag
      601 acccagggct tcgatgaagg catccatgaa gcggacgtgg tcgtcgaaga aataacccag
      661 gtctggtttg tcggatttgc ccataccgat caggtctgga gcaatgcagc gatgggtcgg
      721 tgcaacatgc gggatgatgt tgcgccacac gtaggaggag gtcgggttac cgtgcaggaa
      781 cagcacaggg gtgccatcgc gcggaccaac atcgacgtag tgcatgcgct cgcccaggac
      841 ttccacataa tgggggtcga atggaaagcc agtaccgatt tcggatccat cgttatcgct
      901 ctgaaagtac agatcctcca tgaattccat gttggctgca cagccgccgc cgccgccgcc
      961 gccgccgcgt tcggttcgac gcttcgcggc tgccgctcga cttatgctgc gcttcccgca
     1021 ggaaccgcgc tgccgcccgt gtagccgccc ccgccggcga tcgggaacga ggaaggagga
     1081 caacacagag gagagaatta accgccggta gaaccactgc cagccttcct gcctgacggg
     1141 cgggctacga gtgggaggag ggaagggttc gcgtcgaagg gcgctagggc ggagtcaagg
     1201 gcgtgcgtgt tgattggctt ctcttggtag agagggcgga gtctcgcggg tcggcggaag
     1261 cagcgaggtg aggcaggagc actgcggctg cgggggaggg acgtcatgtg cgctcggaga
     1321 cccaagcgct gggtagctct agagagctag gttggaattt aggcataagg accccacaga
     1381 gtcccaccca ctgtcctggt gctgtgcctg gtccagaacg aactcacacg ggaccaggga
     1441 cttggcttga gaccagagga ctcactgcac accaaccacg ggacccctga gacagacctc
     1501 taagaatacg tggcccacat tgtgcactct gatgaactta gaccatcaga gtgggacaga
     1561 aaccaaaaag cataacccag acccagaaaa aaactttcac cctgtaatga gcataatttt
     1621 aaaaaatgcc ttttagacat ataaaactag attaagcagg aatcccacag catccaaggc
     1681 tgctcctgat ccagagctca gtgacatcac tcacgcaaga taaccagata cagtcctaga
     1741 accccatgat gcccactgga cagaatataa gcagagaatt cgatatcaag cttatcgata
     1801 ccgtcgacct cgaggggggg cccggtaccc agcttttgtt ccctttagtg agggttaatt
     1861 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1921 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1981 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2041 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2101 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2161 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2221 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2281 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2341 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2401 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2461 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2521 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2581 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2641 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2701 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2761 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2821 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2881 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2941 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3001 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3061 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3121 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3181 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3241 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3301 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3361 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3421 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3481 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3541 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3601 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3661 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3721 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3781 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3841 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3901 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3961 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4021 aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4081 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4141 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4201 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4261 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4321 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4381 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4441 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat
     4501 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     4561 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     4621 cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
     4681 tggagctcca ccgcggtggc ggccgctcta gaactagtaa ggctgtcttc tctttcctca
     4741 agatagctaa gttaaattaa actatctcag agaccttcga taaatgaact tgttttagtt
     4801 aaaacaatac acttcaacat tccacatgca ggaaatcctg caagtgatcg tgcaaaaact
     4861 attcttgaac actgaaacgt tatggggaaa cgatcacagt aatcgggcta aaacatatgg
     4921 atacattcac actgttaaga gagtactctt gagggtaacg acgggataat ttaaagtcaa
     4981 attaggacta actccaaata cttcttaata attctagagt gatgctggct tatcctttct
     5041 gaaacacaat cggtatattt aaatccattt tgaaacttca agatttcaat atgtccacaa
     5101 ccaacgctgt gtcattaacg gttaagcccg aggcagaaag catcattagc gctacttaag
     5161 atattttgtg aatgctgttc acgtacacag aagagaagaa aaatattttg gtgctcagca
     5221 cacaagccta ttattccgtc ttgcaaaacc atggccgccg ccaccacgga caagcatcta
     5281 atacaattcc tggctttaat ctaatgtcaa gaggcggtgg ctataatgct ataaattagg
     5341 agacacagtg tacttccgtt cccgtcccga gggagaagcc tcgaaagtac gcgctttaag
     5401 accaagcccc ggagtcttcg agtcctggcc tcaatcgggg ttgcccctcc aaccgcaaac
     5461 gagaagcagc agaggaagac gaggagcaac acctaagcct cgaccggctc caggaccccg
     5521 ccccagggct ccaggagtga aggccaagaa gccaggggtc cggggcgccg cagaacgcga
     5581 ggccaaggcg cgggaagggc cgtgggtgcg tccagatcag ccacaggcag ggggcacagg
     5641 gtgaccgcgt gaggccgggc ctgaaatcca gcgcggatac tcaccaaacc atgccgtagg
     5701 cgccttctcc gatgtacgag aggttggtgt agcgcggccc tacgtcgaac acctgcccgc
     5761 ggaccatctc cgggcccgcc gccgccgccg ccgccatgtt ggctgcacaa ctagtgctac
     5821 cgctgccgct accacc
//
