LOCUS       sequence-47424-e        5973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5973)
  AUTHORS   Zou J, Maeder ML, Mali P, Pruett-Miller SM, Thibodeau-Beganny S, 
            Chou BK, Chen G, Ye Z, Park IH, Daley GQ, Porteus MH, Joung JK, 
            Cheng L
  TITLE     Gene targeting of a disease-related gene in human induced 
            pluripotent stem and embryonic stem cells.
  JOURNAL   Cell Stem Cell. 2009 Jul 2. 5(1):97-110.
  PUBMED    19540188
REFERENCE   2  (bases 1 to 5973)
  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: "Cell Stem 
            Cell. 2009 Jul 2. 5(1):97-110."
COMMENT     Sequence Label: PIGAZFN-L2
FEATURES             Location/Qualifiers
     source          1..5973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             910..930
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             940..963
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1234..1821
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             1837..1878
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1888..1905
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1928..1945)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1934..2158
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2204..2632
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2291..2310)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2501..2522
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2641..2661)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2646..2976
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2827..2962
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2889..2908
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3024..3071
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3090..3488
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3646..3767
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3683..3702)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3737..3756
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3816..3832)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3816..3832)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3829..3851)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3840..3856
                     /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(3864..3870,3871..3888,3889..3894))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3909..3930
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4047..4064)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4218..4806)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4298..4317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4977..5768,5769..5837))
                     /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     5600..5619
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5838..5942)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 accatgggac ctaagaaaaa gaggaaggtg gcggccgctg actacaagga tgacgacgat
      961 aaatctagac ccggggagcg ccccttccag tgtcgcattt gcatgcggaa cttttcggac
     1021 ggcagcacgc ttaacaggca tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg
     1081 atctgtatgc gaaatttctc ccaggggagc agcttgaggc ggcatctacg tacgcacacc
     1141 ggcgagaagc cattccaatg ccgaatatgc atgcgcaact tcagtatcag ccacaacctg
     1201 gcgcggcacc taaaaaccca cctgagggga tcccaactag tcaaaagtga actggaggag
     1261 aagaaatctg aacttcgtca taaattgaaa tatgtgcctc atgaatatat tgaattaatt
     1321 gaaattgcca gaaattccac tcaggataga attcttgaaa tgaaggtaat ggaatttttt
     1381 atgaaagttt atggatatag aggtaaacat ttgggtggat caaggaaacc ggacggagca
     1441 atttatactg tcggatctcc tattgattac ggtgtgatcg tggatactaa agcttatagc
     1501 ggaggttata atctgccaat tggccaagca gatgaaatgc aacgatatgt cgaagaaaat
     1561 caaacacgaa acaaacatat caaccctaat gaatggtgga aagtctatcc atcttctgta
     1621 acggaattta agtttttatt tgtgagtggt cactttaaag gaaactacaa agctcagctt
     1681 acacgattaa atcatatcac taattgtaat ggagctgttc ttagtgtaga agagctttta
     1741 attggtggag aaatgattaa agccggcaca ttaaccttag aggaagtgag acggaaattt
     1801 aataacggcg agataaactt ttaagggccc ttcgaaggta agcctatccc taaccctctc
     1861 ctcggtctcg attctacgcg taccggtcat catcaccatc accattgagt ttaaacccgc
     1921 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1981 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2041 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2101 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2161 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2221 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2281 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2341 cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt acggcacctc
     2401 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2461 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2521 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttggggatt
     2581 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     2641 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccaggcagg cagaagtatg
     2701 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2761 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2821 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2881 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     2941 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3001 attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag
     3061 tataatacga caaggtgagg aactaaacca tggccaagcc tttgtctcaa gaagaatcca
     3121 ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg
     3181 tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt
     3241 ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg
     3301 gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg
     3361 gacggtgtcg acaggtgctt ctcgatctgc atcctgggat caaagcgata gtgaaggaca
     3421 gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg
     3481 agggctaagc acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc
     3541 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3601 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3661 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3721 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3781 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3841 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3901 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3961 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4021 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4081 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4141 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4201 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4261 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4321 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4381 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     4441 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4501 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4561 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4621 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     4681 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4741 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4801 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4861 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4921 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4981 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5041 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5101 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5161 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5221 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5281 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5341 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5401 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5461 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5521 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5581 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5641 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5701 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5761 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5821 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5881 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5941 cgcacatttc cccgaaaagt gccacctgac gtc
//
