LOCUS       sequence-420918-        6461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LR vector for integration of GFP into N2a FRT rtTA3 expression cells
            (BioID vector).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6461)
  AUTHORS   Roth JF, Braunschweig U, Wu M, Li JD, Lin ZY, Larsen B, Weatheritt 
            RJ, Gingras AC, Blencowe BJ
  TITLE     Systematic analysis of alternative exon-dependent interactome 
            remodeling reveals multitasking functions of gene regulatory 
            factors.
  JOURNAL   Mol Cell. 2023 Dec 7;83(23):4222-4238.e10. doi: 
            10.1016/j.molcel.2023.10.034.
  PUBMED    38065061
REFERENCE   2  (bases 1 to 6461)
  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.molcel.2023.10"; journalName: "Mol Cell"; date: 
            "2023-12-7- 7"; volume: "83"; issue: "23"; pages: "4222-4238.e10"
COMMENT     Sequence Label: 5'-BirA-FLAG GFP minimal CMV pCDNA5
FEATURES             Location/Qualifiers
     source          1..6461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(514..561)
                     /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)."
     primer_bind     693..712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    845..1069
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1058..1075
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     protein_bind    1117..1141
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(1143..1856,1857..1859,1860..1862))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1146..1856,1857..1859,1860..1862))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1188..1209)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1537..1556
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1865..1889)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1902..1925)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(join(1956..2564,2565..2567,2568..2918))
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     primer_bind     complement(3051..3075)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3097..3115
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3134..3152
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3171..3189
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3208..3226
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3425..3444
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3519..3623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3624..3692,3693..4484)
                     /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     complement(3842..3861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4655..5243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5144..5163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5397..5414
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5531..5552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5567..5572,5573..5590,5591..5597)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5605..5621
                     /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)."
     primer_bind     5610..5632
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5629..5645
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5629..5645
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5694..5815
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5705..5724)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5759..5778
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5945..6461,1..506))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(join(5945..6461,1..506))
                     /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
                     "
ORIGIN
        1 acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg
       61 aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc
      121 gtctggctaa gatcggccgc agcgatcgca tccatggcct ccgcgaccgg ctgcagaaca
      181 gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg
      241 caataggtca ggctctcgct gaattcccca atgtcaagca cttccggaat cgggagcgcg
      301 gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt
      361 acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc
      421 tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca
      481 gacgtcgcgg tgagttcagg ctttttggcc aaggaagttc ctatactttc tagagaatag
      541 gaacttcgga ataggaactt ctaggtacgt gaaccatcac cctaatcaag ttttttgggg
      601 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
      661 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
      721 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
      781 gcgccgctac agggcgcgtg gggatacccc ctagagcccc agctggttct ttccgcctca
      841 gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc
      901 ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag acaatgcgat
      961 gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa
     1021 ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat
     1081 cagcgggttt aaacgggccc tctagactcg agtcacacca ctttgtacaa gaaagttggg
     1141 tactacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     1201 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg
     1261 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     1321 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     1381 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     1441 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     1501 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     1561 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     1621 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
     1681 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     1741 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     1801 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     1861 atggtgccaa cttttttgta caaacttgtg ggcgcgccta gtttatcgtc atcgtctttg
     1921 tagtctggcg cgccgccgcc ggggccagat ccccccttct cggcgctccg caggctgatc
     1981 tcgccgccca tccagggctt gatgatgccg tcctgctcca gcagcagggc gccctgcttg
     2041 tcgatgcccc ggctgatgcc gaagatctcc ttgtcgccga tgatcagctt cacgggccgg
     2101 ttgatgaagt tgtccagctt ctcccaccgg ctcaggtagg gggccaggcc ctcctgctcg
     2161 aacagctcca gggcggcccg cagctcccgg atcagcatgg cggccagggt gttccggtcc
     2221 aggttgatgc cggcctcctg cagggtgatc cagccctggt tcaccacgct ctcctccacc
     2281 cgccgcatgg ccatgttgat gccggcgccg atcacgatct gggcggcgtc gccggtcttg
     2341 ccggtcagct ccaccaggat gccggccagc ttccggtcct gcaggtacag gtcgttgggc
     2401 cacttcaccc gcaccttgtc ggcgcccagc ttccgcagca cctcggccat cacgatgccg
     2461 atcaccaggc tcaggccgat ggcggcggcg gggccctgct ccagccgcca gaacatgctc
     2521 aggtacaggt tggcgccgaa ggggctgaac cacttccggc cccggccgcc ccggccggcc
     2581 tgctggtact cggcgatgca ggcgtcgccg ctcttcagct cgccgatccg gtccagcagg
     2641 tactggttgg tgctgtcgat cacgggcagc acggccacgc tgccgccgtc cagctggccc
     2701 aggatctgct tggcgttcag cagctggatg ggctcgggca ggctgtagcc cttgccgggc
     2761 acggtgaaca cgtccacgcc ccagtcccgc agggtctgga tgtgcttgtt gatggcggcc
     2821 cggctcatgc ccagggtctc gcccagctgc tcgccgctgt ggaactcgcc gttggccagc
     2881 agggcgatca gcttcagggg cacggtgttg tccttcatgg tggcgctagc tggatccgag
     2941 ctcggtacca agcttaagtt taaacgctag aggatccagc ctccgcggag gctggatcgg
     3001 tcccggtgtc ttctatggag gtcaaaacag cgtggatggc gtctccaggc gatctgacgg
     3061 ttcactaaac gagctctgct tatatagggg gtaccttctc tatcactgat agggagctcg
     3121 agacgtgtcg acttctctat cactgatagg gagacgttct agacgtacct tctctatcac
     3181 tgatagggag ctcgagacgt gtcgacttct ctatcactga tagggagacg tgcggccagc
     3241 ttggatccct cgagacgcgt atatctggcc cgtacatcgc gaagcagcgc aaaacgccta
     3301 accctaagca gattcttcat gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt
     3361 ttgctcgcgc actactcagc gacctccaac acacaagcag ggagcagata ctggcttaac
     3421 tatgcggcat cagagcagat tgtactgaga gtgcaccata ggggatcggg agatctcccg
     3481 atccgtcgac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3541 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3601 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3661 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3721 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3781 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3841 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3901 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3961 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4021 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4081 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4141 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4201 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4261 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4321 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4381 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4441 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4501 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4561 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4801 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5281 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5341 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5401 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5461 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5521 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5581 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     5641 atgaccatga ttacgccaag ctctagctag aggtcgacgg tatacagaca tgataagata
     5701 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     5761 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttggggt
     5821 gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga
     5881 aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtagta
     5941 cgtgctattc ctttgccctc ggacgagtgc tggggcgtcg gtttccacta tcggcgagta
     6001 cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt gtacgcccga
     6061 cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat
     6121 cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg agcatatacg
     6181 cccggagccg cggcgatcct gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct
     6241 gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg tattgggaat
     6301 ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg tccgtcagga
     6361 cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag tcctcggccc
     6421 aaagcatcag ctcatcgaga gcctgcgcga cggacgcact g
//
