LOCUS       sequence-213384-        7097 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for knock-in HaloTag fusing to the c-terminal of mouse
            Cdk9 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7097)
  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 7097)
  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..7097
                     /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             4794..4814
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             4830..5714
                     /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(6838..6857)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6839..6857)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6867..6884)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6867..6883)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6876..6898)
                     /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 caggaacaag gtggggcaga atggggcttc tctgctcttg actgctgtgt aggccaggct
     2701 ccaggatgct atgaaacctg actgagctct tgtgtgtcct cccttcagat cctgcacagg
     2761 gacatgaagg ctgcgaatgt gctcattacc cgggatgggg tcctgaagct ggcagatttt
     2821 gggctggccc gtgccttcag cctagctaag aatagccagc ccaaccgata caccaaccgt
     2881 gtggtgacat tgtggtaccg gcctccggag ctgctgctcg gtgaggactc ccgagcgggc
     2941 agaggggggc aggggccggg tgcctacctg gccacagccc ccattgcctg gggccttttg
     3001 agccatcagc tctgggacgt ggagtctcag gaggcccttg ggcttaagag gcactcccgg
     3061 taccctcttc ttctcaggag agcgggacta cggcccgccc attgaccttt ggggtgctgg
     3121 gtgcatcatg gcagagatgt ggactcgtag ccctatcatg cagggtaaca cagagcagca
     3181 ccagctcgcc ctcatcagcc agctctgtgg ctccatcacc ccagaggtga gtaaccgaac
     3241 tgggtgttcc tgggggatgc agagacccag ggcctctggc cagtagtgga gagtggagtc
     3301 cttctgtaga ggaagctgat tgctgcaggc caggcttact gttcagtttg tttgtttgtt
     3361 tgtttgtttg ggtttttggt ttggtttggt ttggtttggt tttttgagac agggtttctc
     3421 tgcgtagccc tggctgtcct ggaactcact ctgtaggcca ggctggcctc gaactcagaa
     3481 atctacctgc ctctgcctcc caagtgctgg gattaaaggc gtgtgccacc acgcctggca
     3541 tggtgcttac cactcttaat gttggttgta tgagctctct gttgccgaga caaagtaagt
     3601 acatgagaaa aacaaaagcg ccaagagttg cttttgttca gaaaaacaga gaggttgttt
     3661 agcctttgct ttggtgcaga gatgctgcta aatgtcatag tagagagtcc actggagaga
     3721 gggtgtggcc atggcaggga gggagcagaa gagcagggtc cagtgtgtag ctccagtggt
     3781 ggcccactca accccagagg cttcagcaca cattcagcag gcctcatggt tccttcccct
     3841 ttgggtggtc ccctctcctc ccagaagttt ctgccacctc ccaatagcgc cacctgctgg
     3901 agaccaagca tcagcacaag cttctagaga cagagtttct tggttttttt gttttgtttt
     3961 gttttattgg ggtttttttt cttggtattt cgttcgagac agggtttctg tatagccctg
     4021 gctgtcctgg aactcaatgt agaccaggct gacctcgaac tcaggaatcc gcctgcctct
     4081 gcctcccaag tgctgggatt aaaggtgtgc gctaccactg cccagctcgt gtggtttttt
     4141 gtttgtttgt tttgtttatt ttttgtttgt aaaatgggaa tgtgcctgga tgggggctgg
     4201 cacatttatc gcagctgact tgtgttcggg cactaggggt gtgctccatc tggtgactgg
     4261 tgcttttaac ttgaagacat gatctctgcg atggagggga gctcagggaa cctctgagag
     4321 aaattgtttt agaaacccac tcccctggca tctcagatag cagtcttact ccaactccgt
     4381 tctctcctca acgcctctct taggtgtggc caaacgtaga caagtatgag ctgtttgaaa
     4441 agctggaact tgtgaagggc cagaagcgga aggtgaagga ccgtctgaag gcctacgtgc
     4501 gcgaccccta tgctctggac ctcattgaca agctgctggt gctggacccc gcgcagcgga
     4561 ttgacagtga cgatgccctc aaccacgact tcttctggtc ggaccccatg ccctcggacc
     4621 tcaagggcat gctgtctact cacttgacgt ctatgtttga gtacctggca ccgccacggc
     4681 ggaagggcag ccagatcacc cagcagtcca ccaaccagag cagaaatccc gccaccacca
     4741 accagacgga atttgaacgt gtcttcggta gcggcagcgg tagcgaattc atggaggatc
     4801 tgtactttca gagcgataac gatggatccg aaatcggtac tggctttcca ttcgaccccc
     4861 attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg cgcgatggca
     4921 cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc aacatcatcc
     4981 cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg ggcaaatccg
     5041 acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat gccttcatcg
     5101 aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc gctctgggtt
     5161 tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc
     5221 gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc caggccttcc
     5281 gcaccaccga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc gagggtacgc
     5341 tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat ggaccattac cgcgagccgt
     5401 tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc aaacgagctg ccaatcgccg
     5461 gtgagccagc gaacatcgtc gcgctggtcg aagaatacat ggactggctg caccagtccc
     5521 ctgtcccgaa gctgctgttc tggggcaccc caggcgttct gatcccaccg gccgaagccg
     5581 ctcgcctggc caaaagcctg cctaactgca aggctgtgga catcggcccg ggtctgaatc
     5641 tgctgcaaga agacaacccg gacctgatcg gcagcgagat cgcgcgctgg ctgtctactc
     5701 tggagatttc cggtggtagc ggcagcggta gcactagttg agaactggcc ggtgttgttc
     5761 ttaggttagg agtggattgc ttcctttgca tggaacatgt gacatgatgg gacatttgag
     5821 attttctcct ccagtacata ttaactccca ccctggcact gggaacagct gcctgagtgg
     5881 gctggggtgg agctatcctg cctgcccctc tctggaggtc tcagcgtgga cacaaagtga
     5941 ggtgggagag gctcacccac tgtgactttc taagaactac ttgcatggtg ggaggaaggc
     6001 acaggtccct cgccatcgcc agctcttcct ctggatgaga gtttgaggga cagaggcgcc
     6061 tccaaaatgg gctacttttg gtctggacct taggggcgtg gggctgtgtg aactcctggt
     6121 tcctttaaaa ggagaatttt attttgtttt gttaagttta gagacattcc tggacgcagt
     6181 ttgattggtt aaattaaaag ttggttattt ttttccagta aatggctgcg tttcctgtgt
     6241 tcagaccttc cctcagctct tgtctcagct agaagctagg gtttggtcag ccaggacact
     6301 gccagttctt ctgtaccacc caacactctg gagacgttca gggtttgccc tcttctcctc
     6361 ctgggtacct tccactgcgc tcttcacccc cgctgaaggc gtgtccacgg tgccagtgtc
     6421 ctgtgcttgg tgttggcaag tgctgtaagc tgagctggag tgagctgctg gacccgggct
     6481 gctttggctc ctaaatcctc agactggact cataagcagg gcactgcagc cttacagtca
     6541 tggagtgaca agtgtagcct gtagctccag ggcttggggg aagcaggaca gaagtggcca
     6601 gagtggggtc aggtggcatg ggttggggtg agggcaggat ggaaggccag tgtggagatg
     6661 ggatggtcct aaggatgcag ggtagggacc cttagggcac tgacctaggc ggtagattcc
     6721 ccaagagttt cctggagtct tctcacagag ctgcctgaaa aggacagttt tagtccgcta
     6781 gctagctctg ggagactagt tctagagcgg ccgccaccgc ggtggagctc caattcgccc
     6841 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     6901 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     6961 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     7021 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     7081 accgctacac ttgccag
//
