LOCUS       sequence-73762-e        8243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Left TALEN for PRKAG2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8243)
  AUTHORS   Miyaoka Y, Chan AH, Judge LM, Yoo J, Huang M, Nguyen TD, Lizarraga 
            PP, So PL, Conklin BR
  TITLE     Isolation of single-base genome-edited human iPS cells without 
            antibiotic selection.
  JOURNAL   Nat Methods. 2014 Mar;11(3):291-3. doi: 10.1038/nmeth.2840. Epub 
            2014 Feb 9.
  PUBMED    24509632
REFERENCE   2  (bases 1 to 8243)
  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"; doi: 
            "10.1038/nmeth.2840"; journalName: "Nat Methods"; date: "2014-03"; 
            volume: "11"; issue: "3"; pages: "291-3"
COMMENT     Sequence Label: MR015-PRKAG2 Left TALEN
FEATURES             Location/Qualifiers
     source          1..8243
                     /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"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..975
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             982..1002
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3837..3854)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3843..4067
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4113..4541
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4200..4219)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4410..4431
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4550..4570)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4555..4884
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4735..4870
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4797..4816
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4951..5745
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5005..5024)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5615..5634
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5919..6040
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5956..5975)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6010..6029
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6089..6105)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6089..6105)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6102..6124)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6113..6129
                     /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(6137..6143,6144..6161,6162..6167))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6182..6203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6320..6337)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6491..7076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6571..6590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7247..8038,8039..8107))
                     /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     7870..7889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8108..8212)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 gccaccatgg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag
      961 gatgacgatg acaagatggc ccccaagaag aagaggaagg tgggcattca ccgcggggta
     1021 cctatggtgg acttgaggac actcggttat tcgcaacagc aacaggagaa aatcaagcct
     1081 aaggtcagga gcaccgtcgc gcaacaccac gaggcgcttg tggggcatgg cttcactcat
     1141 gcgcatattg tcgcgctttc acagcaccct gcggcgcttg ggacggtggc tgtcaaatac
     1201 caagatatga ttgcggccct gcccgaagcc acgcacgagg caattgtagg ggtcggtaaa
     1261 cagtggtcgg gagcgcgagc acttgaggcg ctgctgactg tggcgggtga gcttaggggg
     1321 cctccgctcc agctcgacac cgggcagctg ctgaagatcg cgaagagagg gggagtaaca
     1381 gcggtagagg cagtgcacgc ctggcgcaat gcgctcaccg gtgcccccct gaacctgacc
     1441 ccggaccaag tggtggctat cgccagccac gatggcggca agcaagcgct cgaaacggtg
     1501 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     1561 atcgccagca acggtggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     1621 ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc
     1681 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     1741 ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa
     1801 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1861 gtggctatcg ccagcaacgg tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1921 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     1981 ggtggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     2041 catggcctga ccccggacca agtggtggct atcgccagca acaatggcgg caagcaagcg
     2101 ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gactccggac
     2161 caagtggtgg ctatcgccag ccacgatggc ggcaagcaag cgctcgaaac ggtgcagcgg
     2221 ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc
     2281 agcaacattg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     2341 caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacaa tggcggcaag
     2401 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     2461 ccggaccaag tggtggctat cgccagcaac aatggcggca agcaagcgct cgaaacggtg
     2521 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     2581 atcgccagca acggtggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     2641 ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc
     2701 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     2761 ctgactccgg accaagtggt ggctatcgcc agccacgatg gcggcaagca agcgctcgaa
     2821 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     2881 gtggctatcg ccagcaacat tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     2941 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     3001 ggtggcggca agcaagcgct cgaaagcatt gtggcccagc tgagccggcc tgatccggcg
     3061 ttggccgcgt tgaccaacga ccatctggtg gcgttggcat gtcttggtgg acgacccgcg
     3121 ctcgatgcag tcaaaaaggg tctgcctcat gctcccgcat tgatcaaaag aaccaaccgg
     3181 cggattcccg agagaacttc ccatcgagtc gcgggatccc agctggtgaa gagcgagctg
     3241 gaggagaaga agtccgagct gcggcacaag ctgaagtacg tgccccacga gtacatcgag
     3301 ctgatcgaga tcgccaggaa cagcacccag gaccgcatcc tggagatgaa ggtgatggag
     3361 ttcttcatga aggtgtacgg ctacagggga aagcacctgg gcggaagcag aaagcctgac
     3421 ggcgccatct atacagtggg cagccccatc gattacggcg tgatcgtgga cacaaaggcc
     3481 tacagcggcg gctacaatct gcctatcggc caggccgacg agatgcagag atacgtggag
     3541 gagaaccaga cccggaataa gcacatcaac cccaacgagt ggtggaaggt gtaccctagc
     3601 agcgtgaccg agttcaagtt cctgttcgtg agcggccact tcaagggcaa ctacaaggcc
     3661 cagctgacca ggctgaacca catcaccaac cgcaatggcg ccgtgctgag cgtggaggag
     3721 ctgctgatcg gcggcgagat gatcaaagcc ggcaccctga cactggagga ggtgcggcgc
     3781 aagttcaaca acggcgagat caacttctga taacttaagt ttaaaccgct gatcagcctc
     3841 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3901 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3961 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4021 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     4081 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     4141 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4201 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4261 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4321 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4381 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4441 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4501 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     4561 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4621 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4681 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4741 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4801 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4861 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     4921 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4981 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     5041 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     5101 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     5161 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     5221 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     5281 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     5341 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     5401 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     5461 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     5521 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5581 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5641 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     5701 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5761 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5821 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5881 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5941 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     6001 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     6061 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     6121 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     6181 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6241 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6301 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6361 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6421 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6481 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6541 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6601 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6661 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6721 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6781 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6841 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6901 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6961 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     7021 gtagcggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     7081 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     7141 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     7201 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     7261 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     7321 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7381 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7441 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7501 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7561 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7621 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7681 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7741 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7801 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7861 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7921 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7981 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     8041 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     8101 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     8161 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     8221 cccgaaaagt gccacctgac gtc
//
