LOCUS       sequence-220588-        7657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for integration at the human ROSA26 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7657)
  AUTHORS   Okumura M, Natsume T, Kanemaki MT, Kiyomitsu T
  TITLE     Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling 
            forces as a multi-arm ensemble.
  JOURNAL   Elife. 2018 May 31;7. pii: 36559. doi: 10.7554/eLife.36559.
  PUBMED    29848445
REFERENCE   2  (bases 1 to 7657)
  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: "Elife."; 
            date: "2018-05-31"; volume: "7. pii: 36559. doi"; pages: " 
            10.7554/eLife.36559"
COMMENT     Sequence Label: pMK247 (hROSA26 CMV-MCS-Hygro)
FEATURES             Location/Qualifiers
     source          1..7657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(155..172)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     382..401
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             600..1619
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             600..1619
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(1929..1946)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1935..2159
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3260..3280)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3260..3278)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3299..3315)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3299..3315)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3312..3334)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3323..3339
                     /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(3347..3353,3354..3371,3372..3377))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3392..3413
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3530..3547)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3701..4289)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3781..3800)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4460..5251,5252..5320))
                     /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     5083..5102
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5321..5425)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5451..5906)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5588..5609)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5800..5819
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6033..6055
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6047..6064
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6048..6064
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6074..6092
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        6594..6973
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6974..7177
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7128..7148
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        7222..7240
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          7333..7562
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     7545..7564
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(7645..7657,1..562,563..565,566..574)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
ORIGIN
        1 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
       61 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
      121 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
      181 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
      241 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
      301 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
      361 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
      421 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
      481 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
      541 ctttgaaaaa cacgatgata agcttgccac aacccgtacc aaagatggat agatccggaa
      601 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
      661 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
      721 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
      781 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
      841 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
      901 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
      961 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     1021 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     1081 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     1141 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     1201 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     1261 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     1321 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     1381 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     1441 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     1501 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     1561 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaataga
     1621 gtagatgccg accgaacaag agctgatttc gagaacgcct cagccagcaa ctcgcgcgag
     1681 cctagcaagg caaatgcgag agaacggcct tacgcttggt ggcacagttc tcgtccacag
     1741 ttcgctaagc tcgctcggct gggtcgcggg agggccggtc gcagtgattc aggcccttct
     1801 ggattgtgtt ggtccccagg gcacgattgt catgcccacg cactcgggtg atctgactga
     1861 tcccgcagat tggagatcgc cgcccgtgcc tgccgattgg gtgcagatct ctagagctcg
     1921 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     1981 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2041 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2101 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2161 ttctgaggcg gaaagaacca gctggggctc gatgagggag ctgcggtgga ggaggtggag
     2221 agaaggccgc acccttctgg gcagggggag gggagtgccg caataccttt atgggagttc
     2281 tctgctgcct cccgtcttgt aaggaccgcc ctgggcctgg aagaagccct ccctcctttc
     2341 ctcctcgcgt gatctcgtca tcgcctccat gtcgagtcgc ttctcgatta tgggcgggat
     2401 tcttttgcct aggcttaagg ggctaacttg gtccctgggc gttgccctgc aggggagtga
     2461 gcagctgtaa gatttgaggg gcgactccga ttagtttatc ttcccacgga ctagagttgg
     2521 tgtcgaggtt attgtaataa gggtggggta gggaaatgga gcttagtcat tcacctgggg
     2581 ctgattttat gcaacgagac tgcggattat cactaatcat ttttggagca tttttctaga
     2641 gacagacata aagcatgatc acctgagttt tataccattt gagacccttg ctgcaccacc
     2701 aaagtgtagc atcaggttaa atcttaatag aaaaatttta gcttttgctt gagaaaccag
     2761 tgcttccctc cctcaccctc tctccccagg ctctctaccc ctttgcatcc ctaccaggca
     2821 tcttagcaac tctcactcat acttgatccc attttccatt tgttgtactt gctcctccag
     2881 tattcagaca tagcactagc tttctccctc tcttgatctt gggtagcctg gtgtctcgcg
     2941 aaaccagaca gattggttcc accacaaatt aaggcttgag ctggggcttg actcttaccc
     3001 agcagtgctt ttattcctcc ctagttcacg ttcttaaatg tttatcttga ttttcatttt
     3061 atcctttttc cttagctggg attctgtccc tgaccgtctt cacagtccag gtgatcttga
     3121 ctactgcttt acagagaatt ggatctgagg ttaggcaaca tctccctttt tcttcctcta
     3181 aatacctctc atttctgttc ttaccagtta gtaactgatc tcagatgcct gtgtgatagc
     3241 tggtacccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     3301 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3361 ggagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3421 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3481 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3541 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3601 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3661 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3721 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3781 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3841 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3901 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3961 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4021 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4081 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4141 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4201 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4261 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4321 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4381 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4441 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4501 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4561 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4621 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4681 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4741 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4801 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4861 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4921 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4981 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5041 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5101 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5161 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5221 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5281 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5341 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5401 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     5461 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5521 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5581 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5641 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5701 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5761 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5821 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5881 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     5941 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     6001 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     6061 cagtgagcgc gcgtaatacg actcactata gggcgaattg gagctcggaa ctctggtggg
     6121 aggggagggt cagtgaaatt ggctctggcg cgggcgtcct cccaccctcc ccttccttcg
     6181 ggggagtcgg tttacccgcc gcctgcttgt cttcgacacc tgattggctg tcgaagctgt
     6241 gggaccgggc ccttgctact ggctcgagtc tcacatgagc gaaaccactg cgcggggcgc
     6301 gggggtggcg gggaggcggg cgttggtacg gtcctccccg aggccgagcg ccgcagtgtc
     6361 tggccccgcg cccctgcgca acgtggcagg aagcgcgcgc tggaggcggg ggcgggctgc
     6421 cggccgagac ttctggatgg cggcggccgc ggctccgccc cgggttccca ccgcctgaag
     6481 ggcgagacaa gcccgacctg ctacaggcac tcgtgggggt gggggaggag cgggggtcgg
     6541 tccggctggt ttgtgggtgg gaggcgcttg ttctccaaaa accggcgcgc gttgacattg
     6601 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     6661 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     6721 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     6781 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     6841 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     6901 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     6961 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     7021 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     7081 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     7141 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
     7201 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag cttggtaccg
     7261 agctcggatc cactagtaac ggccgccagt gtgctggaat taattcgctg tctgcgaggg
     7321 ccagctgttg gggtgagtac tccctctcaa aagcgggcat gacttctgcg ctaagattgt
     7381 cagtttccaa aaacgaggag gatttgatat tcacctggcc cgcggtgatg cctttgaggg
     7441 tggccgcgtc catctggtca gaaaagacaa tctttttgtt gtcaagcttg aggtgtggca
     7501 ggcttgagat ctggccatac acttgagtga caatgacatc cactttgcct ttctctccac
     7561 aggtgtccac tcccaggtcc aactgcaggt cgagcatgca tctagggcgg ccgcactaga
     7621 ggaattccgc ccctctcccc cccccccctc tccctcc
//
