LOCUS       sequence-68905-e        6918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate Compatible TALEN Construct site specific modification of
            genome, shortened C Terminus, Codon Optimized FokI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6918)
  TITLE     Bao Lab Golden Gate plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6918)
  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: 
            "Unpublished"
COMMENT     Sequence Label: TALEN_C18
FEATURES             Location/Qualifiers
     source          1..6918
                     /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"
     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"
     CDS             complement(1159..1734)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TGERIAPGVHCLYRCYSPSLRDLQQLPGVELERRPPKLTRHSQQRLKCSRSRPLFTDPY
                     NCLVRGFGQGRNHILVFDSHRPKRRRVL"
     primer_bind     1584..1606
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1598..1615
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1599..1615
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1625..1643
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1691..1711)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1691..1709)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1730..1746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1754..1770
                     /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(1778..1784,1785..1802,1803..1808))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1823..1844
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1895..2482
                     /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(2512..2529)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2518..2742
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2788..3216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2875..2894)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3085..3106
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3225..3245)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3230..3559
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3410..3545
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3472..3491
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3626..4420
                     /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(3680..3699)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4290..4309
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4594..4715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4631..4650)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4685..4704
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4764..4780)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4788..4804
                     /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(4812..4818,4819..4836,4837..4842))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4857..4878
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4995..5012)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5166..5751)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5246..5265)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5922..6713,6714..6782))
                     /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     6545..6564
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6783..6887)
                     /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 ggagacgggc
     1441 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     1501 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     1561 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
     1621 cgcgtaatac gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcctcca
     1681 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     1741 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     1801 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     1861 tgcccgcttt ccaccggtcg tctccaacgg atcccagctg gtgaagagcg agctggagga
     1921 gaagaagtcc gagctgcggc acaagctgaa gtacgtgccc cacgagtaca tcgagctgat
     1981 cgagatcgcc aggaacagca cccaggaccg catcctggag atgaaggtga tggagttctt
     2041 catgaaggtg tacggctaca ggggaaagca cctgggcgga agcagaaagc ctgacggcgc
     2101 catctataca gtgggcagcc ccatcgatta cggcgtgatc gtggacacaa aggcctacag
     2161 cggcggctac aatctgccta tcggccaggc cgacgagatg cagagatacg tggaggagaa
     2221 ccagacccgg aataagcaca tcaaccccaa cgagtggtgg aaggtgtacc ctagcagcgt
     2281 gaccgagttc aagttcctgt tcgtgagcgg ccacttcaag ggcaactaca aggcccagct
     2341 gaccaggctg aaccacatca ccaactgcaa tggcgccgtg ctgagcgtgg aggagctgct
     2401 gatcggcggc gagatgatca aagccggcac cctgacactg gaggaggtgc ggcgcaagtt
     2461 caacaacggc gagatcaact tctgataact taagtttaaa ccgctgatca gcctcgactg
     2521 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2581 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2641 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2701 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2761 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     2821 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2881 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2941 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3001 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3061 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3121 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3181 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3241 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3301 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3361 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3421 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3481 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3541 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     3601 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3661 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3721 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3781 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     3841 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3901 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3961 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4021 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4081 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4141 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4201 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4261 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4321 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4381 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4441 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4501 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4561 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4621 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4681 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4741 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4801 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4861 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4921 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4981 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5041 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5101 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5161 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5221 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5281 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5341 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5401 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5461 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5521 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5581 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5641 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5701 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5761 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5821 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5881 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5941 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6001 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6061 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6121 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6181 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6241 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6301 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6361 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6421 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6481 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6541 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6601 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6661 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6721 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6781 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6841 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6901 aaagtgccac ctgacgtc
//
