LOCUS       sequence-242564-        8249 bp    DNA     circular SYN 14-APR-2025
DEFINITION  left TALENS for introducing a double strand break into the genomic 
            human SFTPC locus at the ATG start site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8249)
  AUTHORS   Jacob A, Morley M, Hawkins F, McCauley KB, Jean JC, Heins H, Na CL, 
            Weaver TE, Vedaie M, Hurley K, Hinds A, Russo SJ, Kook S, Zacharias 
            W, Ochs M, Traber K, Quinton LJ, Crane A, Davis BR, White FV, 
            Wambach J, Whitsett JA, Cole FS, Morrisey EE, Guttentag SH, Beers 
            MF, Kotton DN
  TITLE     Differentiation of Human Pluripotent Stem Cells into Functional Lung
            Alveolar Epithelial Cells.
  JOURNAL   Cell Stem Cell. 2017 Oct 5;21(4):472-488.e10. doi: 
            10.1016/j.stem.2017.08.014. Epub 2017 Sep 28.
  PUBMED    28965766
REFERENCE   2  (bases 1 to 8249)
  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"; doi: 
            "10.1016/j.stem.2017.08.014"; journalName: "Cell Stem Cell"; date: 
            "2017-10-5- 5"; volume: "21"; issue: "4"; pages: "472-488.e10"
COMMENT     Sequence Label: EF1a-TALEN_NN (SPC left)
FEATURES             Location/Qualifiers
     source          1..8249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1416..1432
                     /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        1496..1707
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1720..1988
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2034..2043
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2043..2108
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2115..2135
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4659..5246
                     /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(5264..5281)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5270..5381
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5499..5519)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5504..5833
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5684..5819
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5746..5765
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5882..6907
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             5882..6907
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    7037..7158
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7074..7093)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7128..7147
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7211..8002,8003..8071))
                     /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     7834..7853
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8072..8163)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8205..8224
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8230..8249
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc
     1441 ttcatgcaca gtggcgttgc gcgaaggatc tgcgatcgct ccggtgcccg tcagtgggca
     1501 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt
     1561 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     1621 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     1681 cgcaacgggt ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca
     1741 cgcgcccgcc gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct
     1801 cccgcctgtg gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg
     1861 ggtccgggcc tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg
     1921 ctttgcctga ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt
     1981 tacagatcca agctgtgacc ggcgcctacg ctagaatacg actcactata ggggccacca
     2041 tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg
     2101 acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg
     2161 tagatttgag aactttggga tattcacagc agcagcagga aaagatcaag cccaaagtga
     2221 ggtcgacagt cgcgcagcat cacgaagcgc tggtgggtca tgggtttaca catgcccaca
     2281 tcgtagcctt gtcgcagcac cctgcagccc ttggcacggt cgccgtcaag taccaggaca
     2341 tgattgcggc gttgccggaa gccacacatg aggcgatcgt cggtgtgggg aaacagtgga
     2401 gcggagcccg agcgcttgag gccctgttga cggtcgcggg agagctgaga gggcctcccc
     2461 ttcagctgga cacgggccag ttgctgaaga tcgcgaagcg gggaggagtc acggcggtcg
     2521 aggcggtgca cgcgtggcgc aatgcgctca cgggagcacc cctcaacctg accccagagc
     2581 aggtcgtggc aattgcgagc aacatcgggg gaaagcaggc actcgaaacc gtccagaggt
     2641 tgctgcctgt gctgtgccaa gcgcacggac ttacgccaga gcaggtcgtg gcaattgcga
     2701 gcaacaacgg gggaaagcag gcactcgaaa ccgtccagag gttgctgcct gtgctgtgcc
     2761 aagcgcacgg actaacccca gagcaggtcg tggcaattgc gagccatgac gggggaaagc
     2821 aggcactcga aaccgtccag aggttgctgc ctgtgctgtg ccaagcgcac gggttgaccc
     2881 cagagcaagt cgtggcaatt gcgagcaaca tcgggggaaa gcaggcactc gaaaccgtcc
     2941 agaggttgct gcctgtgctg tgccaagcgc acggcctgac cccagagcag gtcgtggcaa
     3001 ttgcgagcca tgacggggga aagcaggcac tcgaaaccgt ccagaggttg ctgcctgtgc
     3061 tgtgccaagc gcacggactg acaccagagc aggtcgtggc aattgcgagc catgacgggg
     3121 gaaagcaggc actcgaaacc gtccagaggt tgctgcctgt gctgtgccaa gcgcacggac
     3181 ttacacccga acaagtcgtg gcaattgcga gcaacggagg gggaaagcag gcactcgaaa
     3241 ccgtccagag gttgctgcct gtgctgtgcc aagcgcacgg acttacgcca gagcaggtcg
     3301 tggcaattgc gagcaacaac gggggaaagc aggcactcga aaccgtccag aggttgctgc
     3361 ctgtgctgtg ccaagcgcac ggactaaccc cagagcaggt cgtggcaatt gcgagccatg
     3421 acgggggaaa gcaggcactc gaaaccgtcc agaggttgct gcctgtgctg tgccaagcgc
     3481 acgggttgac cccagagcag gtcgtggcaa ttgcgagcaa catcggggga aagcaggcac
     3541 tcgaaaccgt ccagaggttg ctgcctgtgc tgtgccaagc gcacggcctg accccagagc
     3601 aggtcgtggc aattacgagc aacaacgggg gaaagcaggc actcgaaacc gtccagaggt
     3661 tgctgcctgt gctgtgccaa gcgcacggac tgacaccaga gcaggtcgtg gcaattgcga
     3721 gccatgacgg gggaaagcag gcactcgaaa ccgtccagag gttgctgcct gtgctgtgcc
     3781 aagcgcacgg cctcacccca gagcaggtcg tggcaattgc gagcaacatc gggggaaagc
     3841 aggcactcga aaccgtccag aggttgctgc ctgtgctgtg ccaagcgcac ggacttacgc
     3901 cagagcaggt cgtggcaatt gcgagcaaca tcgggggaaa gcaggcactc gaaaccgtcc
     3961 agaggttgct gcctgtgctg tgccaagcgc acggactaac cccagagcag gtcgtggcaa
     4021 ttgcgagcaa caacggggga aagcaggcac tcgaaaccgt ccagaggttg ctgcctgtgc
     4081 tgtgccaagc gcacgggttg accccagagc aggtcgtggc aattgcgagc aacatcgggg
     4141 gaaagcaggc actcgaaacc gtccagaggt tgctgcctgt gctgtgccaa gcgcacggcc
     4201 tgaccccaga gcaggtcgtg gcaattgcga gcaacggagg gggaaagcag gcactcgaaa
     4261 ccgtccagag gttgctgcct gtgctgtgcc aagcgcacgg actgacacca gagcaggtcg
     4321 tggcaattgc gagcaacaac gggggaaagc aggcactcga aaccgtccag aggttgctgc
     4381 ctgtgctgtg ccaagcgcac ggactcacgc ctgagcaggt agtggctatt gcatccaaca
     4441 acgggggcag acccgcactg gagtcaatcg tggcccagct ttcgaggccg gaccccgcgc
     4501 tggccgcact cactaatgat catcttgtag cgctggcctg cctcggcgga cgacccgcct
     4561 tggatgcggt gaagaagggg ctcccgcacg cgcctgcatt gattaagcgg accaacagaa
     4621 ggattcccga gaggacatca catcgagtgg caggttccca actcgtgaag agtgaacttg
     4681 aggagaaaaa gtcggagctg cggcacaaat tgaaatacgt accgcatgaa tacatcgaac
     4741 ttatcgaaat tgctaggaac tcgactcaag acagaatcct tgagatgaag gtaatggagt
     4801 tctttatgaa ggtttatgga taccgaggga agcatctcgg tggatcacga aaacccgacg
     4861 gagcaatcta tacggtgggg agcccgattg attacggagt gatcgtcgac acgaaagcct
     4921 acagcggtgg gtacaatctt cccatcgggc aggcagatga gatgcaacgt tatgtcgaag
     4981 aaaatcagac caggaacaaa cacatcaatc caaatgagtg gtggaaagtg tatccttcat
     5041 cagtgaccga gtttaagttt ttgtttgtct ctgggcattt caaaggcaac tataaggccc
     5101 agctcacacg gttgaatcac attacgaact gcaatggtgc ggttttgtcc gtagaggaac
     5161 tgctcattgg tggagaaatg atcaaagcgg gaactctgac actggaagaa gtcagacgca
     5221 agtttaacaa tggcgagatc aatttccgct cataaaaaat cagcctcgac tgtgccttct
     5281 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5341 actcccactg tcctttccta ataaaatgag gaaattgcat cacaacactc aaccctatct
     5401 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5461 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     5521 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     5581 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     5641 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     5701 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     5761 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     5821 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
     5881 gatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     5941 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     6001 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     6061 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     6121 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     6181 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     6241 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     6301 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     6361 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     6421 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     6481 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     6541 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     6601 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     6661 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     6721 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     6781 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     6841 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     6901 ggaatagcac gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     6961 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     7021 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     7081 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     7141 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     7201 atcatggtca ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7261 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7321 catctggccc cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat
     7381 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7441 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7501 gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7561 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7621 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7681 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7741 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7801 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7861 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7921 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7981 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8041 taagggcgac acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaagca
     8101 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8161 aaataggggt cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta
     8221 tacctgaata tggctcataa caccccttg
//
