LOCUS       sequence-472726-        7576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for generating Flp-In cell lines allowing dox-inducible 
            mammalian expression of miniTurbo fused to full-length NRF1 isoform 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7576)
  TITLE     A Scalable Exon Deletion Platform Coupled To Single-Cell 
            Transcriptomics Reveals Exons Shaping Gene Expression Dynamics
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7576)
  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            "Unpublished"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-FL
FEATURES             Location/Qualifiers
     source          1..7576
                     /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(3462..3486)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3528..3545)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3534..3758
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3891..3910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4042..4089
                     /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             4097..5119
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4097..5119
                     /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    5249..5370
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5286..5305)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5340..5359
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5419..5435)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5419..5435)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5432..5454)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5443..5459
                     /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(5467..5473,5474..5491,5492..5497))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5512..5533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5650..5667)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5821..6409)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5901..5920)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6580..7371,7372..7440))
                     /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(7441..7545)
                     /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 gggtttcatg gacccaggca ctacggacca tagttaaaaa ctgttataaa
     2761 cagcatgggc gggaagacct tttgtatgcc tttgaagatc agcaaacgca aacacaggcc
     2821 acagccacac atagtatagc tcatcttgta ccatcacaga ctgtagtcca gacttttagt
     2881 aaccctgatg gcactgtctc acttatccag gttggtacgg gggcaacagt agccacattg
     2941 gctgatgctt cagaattgcc aaccacggtc accgttgccc aagtgaatta ttctgccgtg
     3001 gctgatggag aggtggaaca aaattgggcc acgttacagg gaggtgagat gaccatccag
     3061 acgacgcaag catcagaggc cacccaggcg gtggcatcgt tggcagaggc cgcagtggca
     3121 gcttctcagg agatgcagca gggagctaca gtcactatgg cgcttaacag cgaagctgcc
     3181 gcccatgctg tcgccaccct ggctgaggcc accttacaag gtgggggaca gatcgtcttg
     3241 tctggggaaa ccgcagcagc cgtcggagca cttactggag tccaagatgc taatggcctg
     3301 gtccagatcc ctgtgagcat gtaccagact gtggtgacca gcctcgccca gggcaacgga
     3361 ccagtgcagg tggccatggc ccctgtgacc accaggatat cagacagcgc agtcaccatg
     3421 gacggccaag ctgtggaggt ggtgacattg gaacagtgat aacccagctt tcttgtacaa
     3481 agtggtgtga ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3541 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3601 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3661 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3721 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3781 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3841 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3901 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3961 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4021 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     4081 aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     4141 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     4201 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     4261 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     4321 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     4381 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     4441 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     4501 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     4561 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     4621 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     4681 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     4741 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     4801 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     4861 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     4921 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     4981 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     5041 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     5101 tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga
     5161 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5221 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5281 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5341 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5401 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5461 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5521 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5581 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5641 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5701 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5761 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5821 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5881 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5941 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6001 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6061 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6121 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6181 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6241 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6301 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6361 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6421 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6481 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6541 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6601 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6661 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6721 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6781 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6841 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6901 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6961 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7021 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7081 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7141 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7201 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7261 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7321 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7381 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7441 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7501 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7561 agtgccacct gacgtc
//
