LOCUS       sequence-120252-        7348 bp    DNA     circular SYN 14-APR-2025
DEFINITION  donor plasmid for targeting PAX6 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7348)
  AUTHORS   Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, Huang CT, Du Z, 
            Zhang SC
  TITLE     Engineering Human Stem Cell Lines with Inducible Gene Knockout using
            CRISPR/Cas9.
  JOURNAL   Cell Stem Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi: 
            10.1016/j.stem.2015.06.001.
  PUBMED    26145478
REFERENCE   2  (bases 1 to 7348)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Stem 
            Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi: 
            10.1016/j.stem.2015.06.001."
COMMENT     Sequence Label: PAX6 CKO 2/7 Donor plasmid
FEATURES             Location/Qualifiers
     source          1..7348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(16..444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(126..147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     338..357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        471..575
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(576..644,645..1436)
                     /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(794..813)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1607..2195
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2096..2115
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2349..2366
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2483..2504
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2519..2524,2525..2542,2543..2549)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2557..2573
                     /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     2562..2584
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2581..2597
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2581..2597
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2618..2636
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3704..3737
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    4179..4212
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    4243..4276
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4337..4833
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4372..4395)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4766..4785
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4873..5472
                     /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(4873..4892)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5369..5389
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    6029..6062
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     RBS             6506..6514
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(7154..7173)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7155..7173)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7180..7197)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7180..7196)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7189..7211)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
       61 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
      121 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
      181 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
      241 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
      301 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
      361 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
      421 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
      481 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
      541 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
      601 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
      661 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
      721 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
      781 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
      841 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
      901 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
      961 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1021 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1081 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     1141 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1201 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1261 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1321 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1381 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1441 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     1501 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     1561 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1621 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     1681 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     1741 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     1801 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     1861 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     1921 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     1981 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2041 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2101 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2161 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2221 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2281 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2341 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     2401 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2461 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     2521 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     2581 caggaaacag ctatgaccat gattacgcca agctcgaaat taaccctcac taaagggaac
     2641 aaaagctggt acccacgctc accccctcct gttcgtactt tttaaagcag tgaggcgagg
     2701 tagacagggt gtgtcacagt acagttaaag gggtgaagat ctaaacgcca aaagagaagt
     2761 taatcacaat aagtgaggtt tgggataaaa agttgggctt gcccctttca aagtcccaga
     2821 aagctgggag gtagatggag agggggccat tgggaagttt ttttggtgta gggagaggag
     2881 tagaagataa agggtaagca gagtgttggg ttctgggggt cttgtgaagt tccttaagga
     2941 aggagggagt gtggccctgc agccctccca aactgctcca gcctatgctc tccggcacca
     3001 ggaagttcca aggttccctt cccctggtct ccaaacttca ggtattcctc tcccctcaca
     3061 ccccttcaac ctcagctctt ggcctctact ccttactcca ctgttcctcc tgtttccccc
     3121 ttcccctttt cctggttctt tatatttttg caaagtggga tccgaacttg ctagattttc
     3181 caattctccc aagccagacc agagcagcct cttttaaagg atggagactt ctgtggcaga
     3241 tgccgctgaa aatgtgggtg taatgctggg acttagagtt tgatgacagt ttgactgagc
     3301 cctagatgca tgtgtttttc ctgagagtga ggctcagaga gcccatggac gtatgctgtt
     3361 gaaccacagc ttgatatacc tttttctcct tctgttttgt cttaggggga agactttaac
     3421 taggggcgcg cagatgtgtg aggcctttta ttgtgagagt ggacagacat ccgagatttc
     3481 aggcaagttc tgtggtggct gctttgggct caaaacccac aaacagtcaa aacaaactat
     3541 ttcagtatgc ttcttcacta tagttcagat agaagaaaat aaatcaaaat tggggcatcg
     3601 cttctactga ttaaagggtt aactgctaaa gaatgtaggg gaaacagatt ggggatattc
     3661 cagtactttg tttcaagccc caaagggtag attttgtatg cacgaagttc ctattctcta
     3721 gaaagtatag gaacttcgtc gactgcaggg cagagctgag tgaccctcag atcgccccaa
     3781 gaggttgagt ggatcaattc ctaaagaacg gagattctcc tgtcctagcc ccaggtccac
     3841 ctcggttggg agttcaggcc tacctgatgc agctgcccga ggattaactc ctattttctt
     3901 gctaacagag ccccatattc gagccccgtg gaatcccgcg gcccccagcc agagccagca
     3961 tgcagaacag taagtgcctc tggtctttct gggacttcgg gctcggggtg cgcggacccg
     4021 agggtcaggg gaggacacag ggactcgcga cgcccgcgct ggcccggccg ccgaccgact
     4081 gaggcccggg gacctgcggc ggctgctccc cgctccgcgc ccggcagccc gggaggggag
     4141 ggcgtttgct tggctcttct cgatactgga gaagcaggga agttcctatt ctctagaaag
     4201 tataggaact tcgaattccg atcatattca ataaccctta atataacttc gtataatgta
     4261 tgctatacga agttattagg tctgaagagg agtttacgtc cagccaagct taggatctcg
     4321 acctcgaaat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
     4381 ttagcagccc cgctggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     4441 catccaccgg tagcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac
     4501 tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca
     4561 aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa
     4621 gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca
     4681 gcagctggga agggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     4741 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     4801 ctgttctcct cttcctcatc tccgggcctt tcgacctgca tccatctaga tctcgagcag
     4861 ctgaagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     4921 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     4981 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     5041 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     5101 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     5161 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     5221 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     5281 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     5341 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     5401 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     5461 cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc
     5521 catgcatcga tgatatcaga tccccgggat gcagaaattg atgatctatt aaacaataaa
     5581 gatgtccact aaaatggaag tttttcctgt catactttgt taagaagggt gagaacagag
     5641 tacctacatt ttgaatggaa ggattggagc tacgggggtg ggggtggggt gggattagat
     5701 aaatgcctgc tctttactga aggctcttta ctattgcttt atgataatgt ttcatagttg
     5761 gatatcataa tttaaacaag caaaaccaaa ttaagggcca gctcattcct cccactcatg
     5821 atctatagat ctatagatct ctcgtgggat cattgttttt ctcttgattc ccactttgtg
     5881 gttctaagta ctgtggtttc caaatgtgtc agtttcatag cctgaagaac gagatcagca
     5941 gcctctgttc cacatacact tcattctcag tattgttttg ccaagttcta attccatcag
     6001 aagctggtcg agatccggaa cccttaatat aacttcgtat aatgtatgct atacgaagtt
     6061 attaggtccc tcgaagaggt tcactaggat ccgacaagca gaagaacggc atcaggctgg
     6121 gcccttaaga acagcccgcc cgagttttcg aagctgcggt agagaaaccc catccccccc
     6181 aaatcctgta actcacaccc cctctcgccc ctgcccgggc tctgaccccg gagagagggg
     6241 ctgggctgat cagggcgggg cgaagtcttc tcctagttga gcgagaagga ggcccctttg
     6301 ggcttagcca agactgaaag tctccaccaa cccggcgggg ttgggggctt ctctccccaa
     6361 gtcactctca gggcagctcc tcccctcctc accccactac gggcctagaa gggatgggga
     6421 gaggagcccc caggcctgca gcagggtagg ggtggtaaac tcgctgttac agggcgcggg
     6481 agagagcacg gtttactggg tctggtaagg aggtgcccag gaattggaaa atccctgccc
     6541 tggcaactgc aattgcttgg gtcctgatag tcccaggccc agagcgcttt tgatacttat
     6601 ggcccttgta cctccctgct ttcctctgcc cagaccccgt ggggtcccac ccaccttcct
     6661 gttcgtggtg ggtgctggta cgcggcaggg ctagtctaga gctccgacct gggagcctgg
     6721 aggcccagcg aaaccaggcc cggtttgagg ttacggttga ggcacacggc gtcggggctc
     6781 aactgaactc aaagtgtgga aaggcccggg tcttccagca gcctgccctg gccgaaccac
     6841 acaggcttaa ggagggtccc cagcacctta tccatctgtt ttggaaacgc cggtcgcccc
     6901 ctggtgcttc cggggctggt aagcgcgtag ctgaggaaag ggcacaaagg tggcgagctt
     6961 tgcgggagta gttgtgcaga acctgccgga ccccagggct tggccggtag gctcgccttc
     7021 ggcggctggg ctctcccctt ggagctcaga ctccccagcc gcaggctgtc tctcgggcgg
     7081 gccccagcca gggcgcgcag ccctgagcgt ttgtcgctgc gggcggccgc caccgcggtg
     7141 gagctccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg
     7201 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     7261 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     7321 cctgaatggc gaatggaaat tgtaagcg
//
