LOCUS       sequence-354422-        8249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  right 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 Dec 15, 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_HD (SPC right)
FEATURES             Location/Qualifiers
     source          1..8249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             225..493
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      539..548
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             548..613
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             620..640
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3164..3751
                     /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(3769..3786)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3775..3886
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4004..4024)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4009..4338
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4189..4324
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4251..4270
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4387..5412
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4387..5412
                     /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    5542..5663
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5579..5598)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5633..5652
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5716..6507,6508..6576))
                     /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     6339..6358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6577..6668)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6710..6729
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6735..6754
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6840..7428
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7329..7348
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7582..7599
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7697..7714
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7698..7714
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8170..8186
                     /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)."
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
       61 cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac agctgaagct tcgaggggct cgcatctctc
      241 cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt cgcgttctgc
      301 cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca
      361 ggtcgggtcc gggcctttgt ccggcgctcc cttggagcct acctagactc agccggctct
      421 ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt tctgttctgc
      481 gccgttacag atccaagctg tgaccggcgc ctacgctaga atacgactca ctataggggc
      541 caccatggac tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga
      601 cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc
      661 agccgtagat ttgagaactt tgggatattc acagcagcag caggaaaaga tcaagcccaa
      721 agtgaggtcg acagtcgcgc agcatcacga agcgctggtg ggtcatgggt ttacacatgc
      781 ccacatcgta gccttgtcgc agcaccctgc agcccttggc acggtcgccg tcaagtacca
      841 ggacatgatt gcggcgttgc cggaagccac acatgaggcg atcgtcggtg tggggaaaca
      901 gtggagcgga gcccgagcgc ttgaggccct gttgacggtc gcgggagagc tgagagggcc
      961 tccccttcag ctggacacgg gccagttgct gaagatcgcg aagcggggag gagtcacggc
     1021 ggtcgaggcg gtgcacgcgt ggcgcaatgc gctcacggga gcacccctca acctgacccc
     1081 agagcaggtc gtggcaattg cgagccatga cgggggaaag caggcactcg aaaccgtcca
     1141 gaggttgctg cctgtgctgt gccaagcgca cggacttacg ccagagcagg tcgtggcaat
     1201 tgcgagcaac atcgggggaa agcaggcact cgaaaccgtc cagaggttgc tgcctgtgct
     1261 gtgccaagcg cacggactaa ccccagagca ggtcgtggca attgcgagcc atgacggggg
     1321 aaagcaggca ctcgaaaccg tccagaggtt gctgcctgtg ctgtgccaag cgcacgggtt
     1381 gaccccagag caggtcgtgg caattgcgag ccatgacggg ggaaagcagg cactcgaaac
     1441 cgtccagagg ttgctgcctg tgctgtgcca agcgcacggc ctgaccccag agcaggtcgt
     1501 ggcaattgcg agcaacaacg ggggaaagca ggcactcgaa accgtccaga ggttgctgcc
     1561 tgtgctgtgc caagcgcacg gactgacacc agagcaggtc gtggcaattg cgagcaacaa
     1621 cgggggaaag caggcactcg aaaccgtcca gaggttgctg cctgtgctgt gccaagcgca
     1681 cggacttaca cccgaacaag tcgtggcaat tgcgagccat gacgggggaa agcaggcact
     1741 cgaaaccgtc cagaggttgc tgcctgtgct gtgccaagcg cacggactta cgccagagca
     1801 ggtcgtggca attgcgagca acaacggggg aaagcaggca ctcgaaaccg tccagaggtt
     1861 gctgcctgtg ctgtgccaag cgcacggact aaccccagag caggtcgtgg caattgcgag
     1921 caacaacggg ggaaagcagg cactcgaaac cgtccagagg ttgctgcctg tgctgtgcca
     1981 agcgcacggg ttgaccccag agcaggtcgt ggcaattgcg agcaacaacg ggggaaagca
     2041 ggcactcgaa accgtccaga ggttgctgcc tgtgctgtgc caagcgcacg gcctgacccc
     2101 agagcaggtc gtggcaattg cgagccatga cgggggaaag caggcactcg aaaccgtcca
     2161 gaggttgctg cctgtgctgt gccaagcgca cggactgaca ccagagcagg tcgtggcaat
     2221 tgcgagcaac ggagggggaa agcaggcact cgaaaccgtc cagaggttgc tgcctgtgct
     2281 gtgccaagcg cacggcctca ccccagagca ggtcgtggca attgcgagcc atgacggggg
     2341 aaagcaggca ctcgaaaccg tccagaggtt gctgcctgtg ctgtgccaag cgcacggact
     2401 tacgccagag caggtcgtgg caattgcgag caacggaggg ggaaagcagg cactcgaaac
     2461 cgtccagagg ttgctgcctg tgctgtgcca agcgcacgga ctaaccccag agcaggtcgt
     2521 ggcaattgcg agccatgacg ggggaaagca ggcactcgaa accgtccaga ggttgctgcc
     2581 tgtgctgtgc caagcgcacg ggttgacccc agagcaggtc gtggcaattg cgagccatga
     2641 cgggggaaag caggcactcg aaaccgtcca gaggttgctg cctgtgctgt gccaagcgca
     2701 cggcctgacc ccagagcagg tcgtggcaat tgcgagcaac atcgggggaa agcaggcact
     2761 cgaaaccgtc cagaggttgc tgcctgtgct gtgccaagcg cacggactga caccagagca
     2821 ggtcgtggca attgcgagca acggaggggg aaagcaggca ctcgaaaccg tccagaggtt
     2881 gctgcctgtg ctgtgccaag cgcacggact cacgcctgag caggtagtgg ctattgcatc
     2941 ccatgacggg ggcagacccg cactggagtc aatcgtggcc cagctttcga ggccggaccc
     3001 cgcgctggcc gcactcacta atgatcatct tgtagcgctg gcctgcctcg gcggacgacc
     3061 cgccttggat gcggtgaaga aggggctccc gcacgcgcct gcattgatta agcggaccaa
     3121 cagaaggatt cccgagagga catcacatcg agtggcaggt tcccaactcg tgaagagtga
     3181 acttgaggag aaaaagtcgg agctgcggca caaattgaaa tacgtaccgc atgaatacat
     3241 cgaacttatc gaaattgcta ggaactcgac tcaagacaga atccttgaga tgaaggtaat
     3301 ggagttcttt atgaaggttt atggataccg agggaagcat ctcggtggat cacgaaaacc
     3361 cgacggagca atctatacgg tggggagccc gattgattac ggagtgatcg tcgacacgaa
     3421 agcctacagc ggtgggtaca atcttcccat cgggcaggca gatgagatgc aacgttatgt
     3481 cgaagaaaat cagaccagga acaaacacat caatccaaat gagtggtgga aagtgtatcc
     3541 ttcatcagtg accgagttta agtttttgtt tgtctctggg catttcaaag gcaactataa
     3601 ggcccagctc acacggttga atcacattac gaactgcaat ggtgcggttt tgtccgtaga
     3661 ggaactgctc attggtggag aaatgatcaa agcgggaact ctgacactgg aagaagtcag
     3721 acgcaagttt aacaatggcg agatcaattt ccgctcataa aaaatcagcc tcgactgtgc
     3781 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3841 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcacaa cactcaaccc
     3901 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3961 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     4021 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4081 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4141 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4201 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4261 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4321 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag
     4381 cacgtgatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
     4441 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
     4501 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     4561 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     4621 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     4681 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     4741 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     4801 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     4861 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     4921 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     4981 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     5041 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     5101 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     5161 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     5221 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     5281 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     5341 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     5401 gcaaaggaat agcacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5461 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5521 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5581 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5641 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5701 gcgtaatcat ggtcattacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5761 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5821 cttaccatct ggccccagcg ctgcgatgat accgcgagaa ccacgctcac cggctccgga
     5881 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5941 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6001 taatagtttg cgcaacgttg ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt
     6061 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6121 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6181 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6241 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6301 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6361 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6421 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6481 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6541 gggaataagg gcgacacgga aatgttgaat actcatattc ttcctttttc aatattattg
     6601 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6661 taaacaaata ggggtcagtg ttacaaccaa ttaaccaatt ctgaacatta tcgcgagccc
     6721 atttatacct gaatatggct cataacaccc cttgctcatg accaaaatcc cttaacgtga
     6781 gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6841 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6901 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6961 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc ccaccacttc
     7021 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     7081 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7141 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7201 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7261 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7321 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7381 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7441 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7501 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     7561 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg cgagagtagg
     7621 gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt tgcgtttcta
     7681 caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc ctgggcggtt
     7741 ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg cgggtttttt
     7801 tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc tgtcactttg
     7861 cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca ctttaaataa
     7921 gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct attatttatg
     7981 attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat aaatctcgaa
     8041 aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg ataatacaaa
     8101 atataataca aactataaga tgttatcagt atttattatc atttagaata aattttgtgt
     8161 cgcccttaat tgtgagcgga taacaattac gagcttcatg cacagtggcg ttgcgcgaag
     8221 gatctgcgat cgctccggtg cccgtcagt
//
