LOCUS       Exported                7867 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Donor for integration at the human AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    pMK231 (AAVS1 CMV-MCS-PURO)
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
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2018-05-31"; volume: "7. pii: 36559. doi"; pages: " 
            10.7554/eLife.36559"
FEATURES             Location/Qualifiers
     source          1..7867
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        143..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
                     /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(6114..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"