LOCUS       Exported                8141 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses the H11-L2 TALEN in mammalian cells. This TALEN was 
            coexpressed with H11-R2 (plasmid #51555) to cleave the H11 locus..
ACCESSION   .
VERSION     .
KEYWORDS    MR015-H11-L2-TALEN
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8141)
  AUTHORS   Zhu F, Gamboa M, Farruggio AP, Hippenmeyer S, Tasic B, Schule B, 
            Chen-Tsai Y, Calos MP
  TITLE     DICE, an efficient system for iterative genomic editing in human 
            pluripotent stem cells.
  JOURNAL   Nucleic Acids Res. 2013 Dec 4.
  PUBMED    24304893
REFERENCE   2  (bases 1 to 8141)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8141
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     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"
     CDS             3118..3705
                     /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"
     primer_bind     complement(3735..3752)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3741..3965
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4011..4439
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4098..4117)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4308..4329
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4448..4468)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4453..4782
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4633..4768
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4695..4714
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4849..5643
                     /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(4903..4922)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5513..5532
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5817..5938
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5854..5873)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5908..5927
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5987..6003)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5987..6003)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6000..6022)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6011..6027
                     /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(6035..6065)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6080..6101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6218..6235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6389..6974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6469..6488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7145..8005)
                     /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     7768..7787
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8006..8110)
                     /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 cgccagcaac attggcggca agcaagcgct cgaaacggtg
     1501 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     1561 atcgccagca acaatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     1621 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacattggc
     1681 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     1741 ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa
     1801 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1861 gtggctatcg ccagcaacat tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1921 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     1981 ggtggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     2041 catggcctga ctccggacca agtggtggct atcgccagcc acgatggcgg caagcaagcg
     2101 ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gactccggac
     2161 caagtggtgg ctatcgccag ccacgatggc ggcaagcaag cgctcgaaac ggtgcagcgg
     2221 ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc
     2281 agcaacggtg 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 gaccccggac caagtggtgg ctatcgccag caacattggc
     2701 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     2761 ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa
     2821 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     2881 gtggctatcg ccagcaacgg tggcggcaag caagcgctcg aaagcattgt ggcccagctg
     2941 agccggcctg atccggcgtt ggccgcgttg accaacgacc atctggtggc gttggcatgt
     3001 cttggtggac gacccgcgct cgatgcagtc aaaaagggtc tgcctcatgc tcccgcattg
     3061 atcaaaagaa ccaaccggcg gattcccgag agaacttccc atcgagtcgc gggatcccag
     3121 ctggtgaaga gcgagctgga ggagaagaag tccgagctgc ggcacaagct gaagtacgtg
     3181 ccccacgagt acatcgagct gatcgagatc gccaggaaca gcacccagga ccgcatcctg
     3241 gagatgaagg tgatggagtt cttcatgaag gtgtacggct acaggggaaa gcacctgggc
     3301 ggaagcagaa agcctgacgg cgccatctat acagtgggca gccccatcga ttacggcgtg
     3361 atcgtggaca caaaggccta cagcggcggc tacaatctgc ctatcggcca ggccgacgag
     3421 atgcagagat acgtggagga gaaccagacc cggaataagc acatcaaccc caacgagtgg
     3481 tggaaggtgt accctagcag cgtgaccgag ttcaagttcc tgttcgtgag cggccacttc
     3541 aagggcaact acaaggccca gctgaccagg ctgaaccaca tcaccaactg caatggcgcc
     3601 gtgctgagcg tggaggagct gctgatcggc ggcgagatga tcaaagccgg caccctgaca
     3661 ctggaggagg tgcggcgcaa gttcaacaac ggcgagatca acttctgata acttaagttt
     3721 aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3781 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3841 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3901 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3961 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     4021 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4081 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4141 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4201 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4261 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4321 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4381 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     4441 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4501 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4561 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4621 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4681 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     4741 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     4801 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4861 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4921 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4981 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     5041 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     5101 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     5161 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     5221 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     5281 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     5341 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     5401 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     5461 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     5521 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     5581 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5641 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     5701 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5761 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5821 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5881 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5941 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     6001 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     6061 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6121 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6181 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6241 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6301 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6361 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6421 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6481 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6541 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6601 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6661 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6721 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6781 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6841 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6901 gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6961 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     7021 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     7081 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     7141 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     7201 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     7261 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     7321 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7381 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7441 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7501 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7561 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7621 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7681 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7741 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7801 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7861 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7921 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7981 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     8041 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     8101 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
