LOCUS       sequence-202460-        6820 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses TALE-VEGFA-TS3 fused to FRB in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6820)
  AUTHORS   Scot Wolfe
  TITLE     Inducible Cas9-DNA-binding-domain fusions for precise genome editing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6820)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCSD_FRB-TALETS3-3xFLAG-2xNLS
FEATURES             Location/Qualifiers
     source          1..6820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             109..387
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             2629..2694
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2728..2754
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             2800..2820
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2865..2999
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2889..2908)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2943..2962
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3116..3136)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3116..3134)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3155..3171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3155..3171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3168..3190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3179..3195
                     /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(3203..3209,3210..3227,3228..3233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3248..3269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3386..3403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3557..4145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3637..3656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4316..5107,5108..5176))
                     /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     4939..4958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5177..5281)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5307..5762)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5444..5465)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5656..5675
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5836..6820
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atctgccacc atgggttcgg gccgtacgat cctctggcat
      121 gagatgtggc atgaaggcct ggaagaggca tctcgtttgt actttgggga aaggaacgtg
      181 aaaggcatgt ttgaggtgct ggagcccttg catgctatga tggaacgggg cccccagact
      241 ctgaaggaaa catcctttaa tcaggcctat ggtcgagatt taatggaggc ccaagagtgg
      301 tgcaggaagt acatgaaatc agggaatgtc aaggacctca cccaagcctg ggacctctat
      361 tatcatgtgt tccgacgaat ctcaaagggc gccggcggaa gcggggtacc tatggtggac
      421 ttgaggacac tcggttattc gcaacagcaa caggagaaaa tcaagcctaa ggtcaggagc
      481 accgtcgcgc aacaccacga ggcgcttgtg gggcatggct tcactcatgc gcatattgtc
      541 gcgctttcac agcaccctgc ggcgcttggg acggtggctg tcaaatacca agatatgatt
      601 gcggccctgc ccgaagccac gcacgaggca attgtagggg tcggtaaaca gtggtcggga
      661 gcgcgagcac ttgaggcgct gctgactgtg gcgggtgagc ttagggggcc tccgctccag
      721 ctcgacaccg ggcagctgct gaagatcgcg aagagagggg gagtaacagc ggtagaggca
      781 gtgcacgcct ggcgcaatgc gctcaccggg gcccccttgc ccctgaacct gaccccggac
      841 caagtggtgg ctatcgccag caacggtggc ggcaagcaag cgctcgaaac ggtgcagcgg
      901 ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc
      961 agcaacaatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     1021 caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacaa tggcggcaag
     1081 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     1141 ccggaccaag tggtggctat cgccagcaac attggcggca agcaagcgct cgaaacggtg
     1201 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     1261 atcgccagca acaatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     1321 ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc
     1381 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     1441 ctgaccccgg accaagtggt ggctatcgcc agcaacaatg gcggcaagca agcgctcgaa
     1501 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1561 gtggctatcg ccagcaacaa tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1621 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     1681 aatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     1741 catggcctga ccccggacca agtggtggct atcgccagca acaatggcgg caagcaagcg
     1801 ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac
     1861 caagtggtgg ctatcgccag caacattggc ggcaagcaag cgctcgaaac ggtgcagcgg
     1921 ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc
     1981 agcaacaatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     2041 caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacat tggcggcaag
     2101 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     2161 ccggaccaag tggtggctat cgccagcaac attggcggca agcaagcgct cgaaacggtg
     2221 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     2281 atcgccagca acaatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     2341 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgctaa taataacgga
     2401 ggacggccag ccttggagtc catcgtagcc caattgtcca ggcccgatcc cgcgttggct
     2461 gcgttaacga atgaccatct ggtggcgttg gcatgtcttg gtggacgacc cgcgctcgat
     2521 gcagtcaaaa agggtctgcc tcatgctccc gcattgatca aaagaaccaa ccggcggatt
     2581 cccgagagaa cttcccatcg agtcgcggga tccgggtcgc tcgagggaga ctacaaagac
     2641 catgacggtg attataaaga tcatgacatc gattacaagg atgacgatga caagcagggg
     2701 tccgagctcg gatcgggtag agccgcgccg gcagctaaga aaaagaaact ggatcaattg
     2761 ggcgctagcg gtagttctgg aggctcaagt ggcgccgctc caaagaagaa gcgtaaggta
     2821 tctgaattct gataatctag aactatagtg agtcgtatta cgtagatcca gacatgataa
     2881 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     2941 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     3001 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     3061 aattcgcggc cgcggcgcca atgcattggg cccggtacgt acccagcttt tgttcccttt
     3121 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3181 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3241 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     3301 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3361 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3421 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3481 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3541 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3601 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3661 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3721 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3781 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3841 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3901 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3961 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     4021 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4081 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4141 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4201 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4261 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4321 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4381 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4441 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4501 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4561 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4621 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4681 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4741 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4801 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4861 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4921 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4981 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5041 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5101 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5161 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5221 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5281 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     5341 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     5401 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     5461 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     5521 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     5581 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     5641 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     5701 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     5761 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     5821 gctattacgc cagtcgacca tagccaattc aatatggcgt atatggactc atgccaattc
     5881 aatatggtgg atctggacct gtgccaattc aatatggcgt atatggactc gtgccaattc
     5941 aatatggtgg atctggaccc cagccaattc aatatggcgg acttggcacc atgccaattc
     6001 aatatggcgg acttggcact gtgccaactg gggaggggtc tacttggcac ggtgccaagt
     6061 ttgaggaggg gtcttggccc tgtgccaagt ccgccatatt gaattggcat ggtgccaata
     6121 atggcggcca tattggctat atgccaggat caatatatag gcaatatcca atatggccct
     6181 atgccaatat ggctattggc caggttcaat actatgtatt ggccctatgc catatagtat
     6241 tccatatatg ggttttccta ttgacgtaga tagcccctcc caatgggcgg tcccatatac
     6301 catatatggg gcttcctaat accgcccata gccactcccc cattgacgtc aatggtctct
     6361 atatatggtc tttcctattg acgtcatatg ggcggtccta ttgacgtata tggcgcctcc
     6421 cccattgacg tcaattacgg taaatggccc gcctggctca atgcccattg acgtcaatag
     6481 gaccacccac cattgacgtc aatgggatgg ctcattgccc attcatatcc gttctcacgc
     6541 cccctattga cgtcaatgac ggtaaatggc ccacttggca gtacatcaat atctattaat
     6601 agtaacttgg caagtacatt actattggaa ggacgccagg gtacattggc agtactccca
     6661 ttgacgtcaa tggcggtaaa tggcccgcga tggctgccaa gtacatcccc attgacgtca
     6721 atggggaggg gcaatgacgc aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc
     6781 ctaatgggag gtctatataa gcaatgctcg tttagggaac
//
