LOCUS       sequence-218263-        7867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for integration at the human AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7867)
  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 7867)
  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: pMK231 (AAVS1 CMV-MCS-PURO)
FEATURES             Location/Qualifiers
     source          1..7867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    295..1098
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     enhancer        1155..1458
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1459..1662
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1613..1633
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    1703..1759
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1884..2005
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1921..1940)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1975..1994
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2014..2514
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2433..2451
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2536..3135
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2536..2555)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3032..3052
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3142..3366
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3369..4205
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(4239..4258)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4240..4258)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4265..4282)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4265..4281)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4274..4296)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             4419..4721
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4600..4619
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             5070..5864
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5070..5864
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5124..5143)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5734..5753
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(6114..6905,6906..6974))
                     /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     6737..6756
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7098..7686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7587..7606
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7840..7857
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc cctttgcttt
      301 ctctgaccag cattctctcc cctgggcctg tgccgctttc tgtctgcagc ttgtggcctg
      361 ggtcacctct acggctggcc cagatccttc cctgccgcct ccttcaggtt ccgtcttcct
      421 ccactccctc ttccccttgc tctctgctgt gttgctgccc aaggatgctc tttccggagc
      481 acttccttct cggcgctgca ccacgtgatg tcctctgagc ggatcctccc cgtgtctggg
      541 tcctctccgg gcatctctcc tccctcaccc aaccccatgc cgtcttcact cgctgggttc
      601 ccttttcctt ctccttctgg ggcctgtgcc atctctcgtt tcttaggatg gccttctccg
      661 acggatgtct cccttgcgtc ccgcctcccc ttcttgtagg cctgcatcat caccgttttt
      721 ctggacaacc ccaaagtacc ccgtctccct ggctttagcc acctctccat cctcttgctt
      781 tctttgcctg gacaccccgt tctcctgtgg attcgggtca cctctcactc ctttcatttg
      841 ggcagctccc ctacccccct tacctctcta gtctgtgcta gctcttccag ccccctgtca
      901 tggcatcttc caggggtccg agagctcagc tagtcttctt cctccaaccc gggcccctat
      961 gtccacttca ggacagcatg tttgctgcct ccagggatcc tgtgtccccg agctgggacc
     1021 accttatatt cccagggccg gttaatgtgg ctctggttct gggtactttt atctgtcccc
     1081 tccaccccac agtggggcaa ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     1141 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     1201 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     1261 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     1321 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     1381 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     1441 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     1501 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     1561 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     1621 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     1681 atccgctagc gctaccggac tcagatctcg agctcaagct tcgaattctg cagtcgacgg
     1741 taccgcgggc ccgggatcca ccggggccgc gactctagat cataatcagc cataccacat
     1801 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     1861 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     1921 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     1981 tgtccaaact catcaatgta tcttaagttg gggttgcgcc ttttccaagg cagccctggg
     2041 tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct
     2101 gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt
     2161 gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc
     2221 gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga
     2281 cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc
     2341 tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg
     2401 gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag
     2461 cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggat
     2521 ccaccggagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     2581 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     2641 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     2701 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     2761 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     2821 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     2881 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     2941 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     3001 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     3061 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     3121 aagcccggtg cctgaggtac cctgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3181 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3241 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3301 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3361 ctatggttac tagggacagg attggtgaca gaaaagcccc atccttaggc ctcctccttc
     3421 ctagtctcct gatattgggt ctaaccccca cctcctgtta ggcagattcc ttatctggtg
     3481 acacaccccc atttcctgga gccatctctc tccttgccag aacctctaag gtttgcttac
     3541 gatggagcca gagaggatcc tgggagggag agcttggcag ggggtgggag ggaagggggg
     3601 gatgcgtgac ctgcccggtt ctcagtggcc accctgcgct accctctccc agaacctgag
     3661 ctgctctgac gcggctgtct ggtgcgtttc actgatcctg gtgctgcagc ttccttacac
     3721 ttcccaagag gagaagcagt ttggaaaaac aaaatcagaa taagttggtc ctgagttcta
     3781 actttggctc ttcacctttc tagtccccaa tttatattgt tcctccgtgc gtcagtttta
     3841 cctgtgagat aaggccagta gccagccccg tcctggcagg gctgtggtga ggaggggggt
     3901 gtccgtgtgg aaaactccct ttgtgagaat ggtgcgtcct aggtgttcac caggtcgtgg
     3961 ccgcctctac tccctttctc tttctccatc cttctttcct taaagagtcc ccagtgctat
     4021 ctgggacata ttcctccgcc cagagcaggg tcccgcttcc ctaaggccct gctctgggct
     4081 tctgggtttg agtccttggc aagcccagga gaggcgctca ggcttccctg tcccccttcc
     4141 tcgtccacca tctcatgccc ctggctctcc tgccccttcc ctacaggggt tcctggctct
     4201 gctctaaggg cgaattcgcg gccgctaaat tcaattcgcc ctatagtgag tcgtattaca
     4261 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4321 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     4381 atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt tacacctata
     4441 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgccgg
     4501 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     4561 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     4621 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     4681 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc atgagattat
     4741 caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag aaacggtgct
     4801 gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga
     4861 gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga
     4921 cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca
     4981 aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagctctg
     5041 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     5101 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     5161 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     5221 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     5281 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     5341 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     5401 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     5461 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     5521 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     5581 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     5641 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     5701 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     5761 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     5821 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt aacgcttaca
     5881 atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatcaggt
     5941 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6001 aatatgtatc cgctcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6061 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6121 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6181 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6241 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6301 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6361 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6421 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6481 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6541 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6601 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6661 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6721 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6781 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6841 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6901 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6961 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7021 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7081 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7141 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7201 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     7261 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7321 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7381 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7441 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7501 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7561 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7621 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7681 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7741 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     7801 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     7861 gcggaag
//
