LOCUS       sequence-472733-        7378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for generating Flp-In cell lines allowing dox-inducible 
            mammalian expression of miniTurbo fused to NRF1 isoform lacking the 
            alternative exon-7 (∆ex7).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7378)
  TITLE     A Scalable Exon Deletion Platform Coupled To Single-Cell 
            Transcriptomics Reveals Exons Shaping Gene Expression Dynamics
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7378)
  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            "Unpublished"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: pcDNA5-miniTurbo-NRF1-∆ex7
FEATURES             Location/Qualifiers
     source          1..7378
                     /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"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1009..1018
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1021..1791
                     /codon_start=1
                     /product="N-terminally truncated mutant of the E. coli 
                     biotin ligase BioID engineered for improved catalytic 
                     efficiency (Branon et al., 2018)"
                     /label=miniTurbo
                     /translation="IPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACI
                     AEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK
                     VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQ
                     EAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIF
                     GISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             1834..1899
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1924..1948
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3264..3288)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3330..3347)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3336..3560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3693..3712)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3844..3891
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3899..4921
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3899..4921
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5051..5172
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5088..5107)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5142..5161
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5221..5237)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5221..5237)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5234..5256)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5245..5261
                     /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(5269..5275,5276..5293,5294..5299))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5314..5335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5452..5469)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5623..6211)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5703..5722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6382..7173,7174..7242))
                     /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"
     promoter        complement(7243..7347)
                     /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 ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cagctagcgc caccatggcg
     1021 atcccgctgc tgaacgctaa acagattctg ggacagctgg acggcgggag cgtggcagtc
     1081 ctgcctgtgg tcgactccac caatcagtac ctgctggatc gaatcggcga gctgaagagt
     1141 ggggatgctt gcattgcaga atatcagcag gcagggagag gaagcagagg gaggaaatgg
     1201 ttctctcctt ttggagctaa cctgtacctg agtatgtttt ggcgcctgaa gcggggacca
     1261 gcagcaatcg gcctgggccc ggtcatcgga attgtcatgg cagaagcgct gcgaaagctg
     1321 ggagcagaca aggtgcgagt caaatggccc aatgacctgt atctgcagga tagaaagctg
     1381 gcaggcatcc tggtggagct ggccggaata acaggcgatg ctgcacagat cgtcattggc
     1441 gccgggatta acgtggctat gaggcgcgtg gaggaaagcg tggtcaatca gggctggatc
     1501 acactgcagg aagcagggat taacctggac aggaatactc tggccgctat gctgatccga
     1561 gagctgcggg cagccctgga actgttcgag caggaaggcc tggctccata tctgtcacgg
     1621 tgggagaagc tggataactt catcaataga cccgtgaagc tgatcattgg ggacaaagag
     1681 attttcggga ttagccgggg gattgataaa cagggagccc tgctgctgga acaggacgga
     1741 gttatcaaac cctggatggg cggagaaatc agtctgcggt ctgccgaaaa gatgcatggg
     1801 ggagggggag gatctggggg agggggagga tctgactaca aggaccacga cggtgactac
     1861 aaggaccacg acatcgacta caaggacgac gacgacaagt taattaaggg cgcgccacct
     1921 aggacaagtt tgtacaaaaa agcaggcttc gaggaacacg gagtgaccca aaccgaacat
     1981 atggctacca tagaagcaca tgcagtggcc cagcaagtgc agcaggtcca tgtggctact
     2041 tacaccgagc atagtatgct gagtgctgat gaagactcgc cttcttctcc cgaggacacc
     2101 tcttacgatg actcagatat actcaactcc acagcagctg atgaggtgac agctcatctg
     2161 gcagctgcag gtcctgtggg aatggccgct gctgctgctg tggcaacagg aaagaaacgg
     2221 aaacggcctc atgtatttga gtctaatcca tctatccgga agaggcaaca aacacgtttg
     2281 cttcggaaac ttcgagccac gttagatgaa tatactactc gtgtgggaca gcaagctatt
     2341 gtcctctgta tctcaccctc caaacctaac cctgtcttta aagtgtttgg tgcagcacct
     2401 ttggagaatg tggtgcgtaa gtacaagagc atgatcctgg aagacctgga gtctgctctg
     2461 gcagaacacg cccctgcgcc acaggaggtt aactcagaac tgccgcctct caccatcgac
     2521 ggaattccag tctctgtgga caaaatgacc caggcccagc ttcgggcatt tatcccagag
     2581 atgctcaagt actctacagg tcggggaaaa ccaggctggg ggaaagaaag ctgcaagccc
     2641 atctggtggc ctgaagatat cccctgggca aatgtccgga gtgatgtccg cacagaagag
     2701 caaaagcaga gggttggtac gggggcaaca gtagccacat tggctgatgc ttcagaattg
     2761 ccaaccacgg tcaccgttgc ccaagtgaat tattctgccg tggctgatgg agaggtggaa
     2821 caaaattggg ccacgttaca gggaggtgag atgaccatcc agacgacgca agcatcagag
     2881 gccacccagg cggtggcatc gttggcagag gccgcagtgg cagcttctca ggagatgcag
     2941 cagggagcta cagtcactat ggcgcttaac agcgaagctg ccgcccatgc tgtcgccacc
     3001 ctggctgagg ccaccttaca aggtggggga cagatcgtct tgtctgggga aaccgcagca
     3061 gccgtcggag cacttactgg agtccaagat gctaatggcc tggtccagat ccctgtgagc
     3121 atgtaccaga ctgtggtgac cagcctcgcc cagggcaacg gaccagtgca ggtggccatg
     3181 gcccctgtga ccaccaggat atcagacagc gcagtcacca tggacggcca agctgtggag
     3241 gtggtgacat tggaacagtg ataacccagc tttcttgtac aaagtggtgt gactcgagtc
     3301 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3361 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3421 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3481 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3541 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3601 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3661 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3721 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3781 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     3841 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     3901 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     3961 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     4021 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     4081 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     4141 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     4201 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     4261 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     4321 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     4381 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     4441 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     4501 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     4561 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     4621 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     4681 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     4741 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     4801 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     4861 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     4921 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4981 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5041 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5101 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5161 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5221 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5281 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5341 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5401 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5461 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5521 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5581 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5641 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5701 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5761 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5821 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5881 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5941 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6001 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6061 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6121 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6181 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6241 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6301 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6361 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6421 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6481 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6541 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6601 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6661 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6721 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6781 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6841 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6901 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6961 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7021 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7081 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7141 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7201 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7261 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7321 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
