LOCUS       sequence-217822-        7096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for knock-in HaloTag fusing to the c-terminal of mouse
            Cdk9 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7096)
  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 7096)
  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: Cdk9-Halo donor
FEATURES             Location/Qualifiers
     source          1..7096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             600..620
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             636..1520
                     /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(2644..2663)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2645..2663)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2673..2690)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2673..2689)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2682..2704)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2830..3285)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2967..2988)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3179..3198
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3312..3416
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3417..3485,3486..4277)
                     /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(3635..3654)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4448..5036
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4937..4956
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5190..5207
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5324..5345
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5360..5365,5366..5383,5384..5390)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5398..5414
                     /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     5403..5425
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5422..5438
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5422..5438
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5457..5477
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5459..5477
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     5506..5522
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5507..5523
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ggctggcaca tttatcgcag ctgacttgtg ttcgggcact aggggtgtgc tccatctggt
       61 gactggtgct tttaacttga agacatgatc tctgcgatgg aggggagctc agggaacctc
      121 tgagagaaat tgttttagaa acccactccc ctggcatctc agatagcagt cttactccaa
      181 ctccgttctc tcctcaacgc ctctcttagg tgtggccaaa cgtagacaag tatgagctgt
      241 ttgaaaagct ggaacttgtg aagggccaga agcggaaggt gaaggaccgt ctgaaggcct
      301 acgtgcgcga cccctatgct ctggacctca ttgacaagct gctggtgctg gaccccgcgc
      361 agcggattga cagtgacgat gccctcaacc acgacttctt ctggtcggac cccatgccct
      421 cggacctcaa gggcatgctg tctactcact tgacgtctat gtttgagtac ctggcaccgc
      481 cacggcggaa gggcagccag atcacccagc agtccaccaa ccagagcaga aatcccgcca
      541 ccaccaacca gacggaattt gaacgtgtct tcggtagcgg cagcggtagc gaattcatgg
      601 aggatctgta ctttcagagc gataacgatg gatccgaaat cggtactggc tttccattcg
      661 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
      721 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
      781 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
      841 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
      901 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
      961 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     1021 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     1081 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     1141 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     1201 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     1261 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     1321 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     1381 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     1441 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     1501 ctactctgga gatttccggt ggtagcggca gcggtagcac tagttgagaa ctggccggtg
     1561 ttgttcttag gttaggagtg gattgcttcc tttgcatgga acatgtgaca tgatgggaca
     1621 tttgagattt tctcctccag tacatattaa ctcccaccct ggcactggga acagctgcct
     1681 gagtgggctg gggtggagct atcctgcctg cccctctctg gaggtctcag cgtggacaca
     1741 aagtgaggtg ggagaggctc acccactgtg actttctaag aactacttgc atggtgggag
     1801 gaaggcacag gtccctcgcc atcgccagct cttcctctgg atgagagttt gagggacaga
     1861 ggcgcctcca aaatgggcta cttttggtct ggaccttagg ggcgtggggc tgtgtgaact
     1921 cctggttcct ttaaaaggag aattttattt tgttttgtta agtttagaga cattcctgga
     1981 cgcagtttga ttggttaaat taaaagttgg ttattttttt ccagtaaatg gctgcgtttc
     2041 ctgtgttcag accttccctc agctcttgtc tcagctagaa gctagggttt ggtcagccag
     2101 gacactgcca gttcttctgt accacccaac actctggaga cgttcagggt ttgccctctt
     2161 ctcctcctgg gtaccttcca ctgcgctctt cacccccgct gaaggcgtgt ccacggtgcc
     2221 agtgtcctgt gcttggtgtt ggcaagtgct gtaagctgag ctggagtgag ctgctggacc
     2281 cgggctgctt tggctcctaa atcctcagac tggactcata agcagggcac tgcagcctta
     2341 cagtcatgga gtgacaagtg tagcctgtag ctccagggct tgggggaagc aggacagaag
     2401 tggccagagt ggggtcaggt ggcatgggtt ggggtgaggg caggatggaa ggccagtgtg
     2461 gagatgggat ggtcctaagg atgcagggta gggaccctta gggcactgac ctaggcggta
     2521 gattccccaa gagtttcctg gagtcttctc acagagctgc ctgaaaagga cagttttagt
     2581 ccgctagcta gctctgggag actagttcta gagcggccgc caccgcggtg gagctccaat
     2641 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     2701 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2761 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2821 ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2881 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2941 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3001 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3061 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3121 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3181 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3241 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     3301 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3361 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3421 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3481 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3541 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3601 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3661 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3721 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3781 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3841 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3901 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3961 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4021 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4081 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4141 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4201 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4261 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4321 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4381 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4441 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4501 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4561 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4621 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4681 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4741 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4801 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4861 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4921 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4981 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5041 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5101 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5161 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     5221 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5281 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     5341 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     5401 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
     5461 ttaaccctca ctaaagggaa caaaagctgg gtaccgggcc ccccctcgag gtcgacggta
     5521 tcgataagct tgatatcgaa ttcaggaaca aggtggggca gaatggggct tctctgctct
     5581 tgactgctgt gtaggccagg ctccaggatg ctatgaaacc tgactgagct cttgtgtgtc
     5641 ctcccttcag atcctgcaca gggacatgaa ggctgcgaat gtgctcatta cccgggatgg
     5701 ggtcctgaag ctggcagatt ttgggctggc ccgtgccttc agcctagcta agaatagcca
     5761 gcccaaccga tacaccaacc gtgtggtgac attgtggtac cggcctccgg agctgctgct
     5821 cggtgaggac tcccgagcgg gcagaggggg gcaggggccg ggtgcctacc tggccacagc
     5881 ccccattgcc tggggccttt tgagccatca gctctgggac gtggagtctc aggaggccct
     5941 tgggcttaag aggcactccc ggtaccctct tcttctcagg agagcgggac tacggcccgc
     6001 ccattgacct ttggggtgct gggtgcatca tggcagagat gtggactcgt agccctatca
     6061 tgcagggtaa cacagagcag caccagctcg ccctcatcag ccagctctgt ggctccatca
     6121 ccccagaggt gagtaaccga actgggtgtt cctgggggat gcagagaccc agggcctctg
     6181 gccagtagtg gagagtggag tccttctgta gaggaagctg attgctgcag gccaggctta
     6241 ctgttcagtt tgtttgtttg tttgtttgtt tgggtttttg gtttggtttg gtttggtttg
     6301 gttttttgag acagggtttc tctgcgtagc cctggctgtc ctggaactca ctctgtaggc
     6361 caggctggcc tcgaactcag aaatctacct gcctctgcct cccaagtgct gggattaaag
     6421 gcgtgtgcca ccacgcctgg catggtgctt accactctta atgttggttg tatgagctct
     6481 ctgttgccga gacaaagtaa gtacatgaga aaaacaaaag cgccaagagt tgcttttgtt
     6541 cagaaaaaca gagaggttgt ttagcctttg ctttggtgca gagatgctgc taaatgtcat
     6601 agtagagagt ccactggaga gagggtgtgg ccatggcagg gagggagcag aagagcaggg
     6661 tccagtgtgt agctccagtg gtggcccact caaccccaga ggcttcagca cacattcagc
     6721 aggcctcatg gttccttccc ctttgggtgg tcccctctcc tcccagaagt ttctgccacc
     6781 tcccaatagc gccacctgct ggagaccaag catcagcaca agcttctaga gacagagttt
     6841 cttggttttt ttgttttgtt ttgttttatt ggggtttttt ttcttggtat ttcgttcgag
     6901 acagggtttc tgtatagccc tggctgtcct ggaactcaat gtagaccagg ctgacctcga
     6961 actcaggaat ccgcctgcct ctgcctccca agtgctggga ttaaaggtgt gcgctaccac
     7021 tgcccagctc gtgtggtttt ttgtttgttt gttttgttta ttttttgttt gtaaaatggg
     7081 aatgtgcctg gatggg
//
