LOCUS       sequence-421248-        7362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LR destination vector for integration into N2a rtTA3 expressing 
            cells with an FRT site in the Rosa26 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7362)
  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 7362)
  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: 3'-BirA*-FLAG minimal CMV pCDNA5
FEATURES             Location/Qualifiers
     source          1..7362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    957..1078
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(994..1013)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1048..1067
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1127..1143)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1127..1143)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1140..1162)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1151..1167
                     /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(1175..1181,1182..1199,1200..1205))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1220..1241
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1358..1375)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1529..2117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1609..1628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2288..3079,3080..3148))
                     /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     2911..2930
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3149..3253)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3328..3347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    3546..3564
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3583..3601
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3620..3638
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3657..3675
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3697..3721
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3826..3950
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3975..3980,3981..3998,3999..4005)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             4059..4718
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4125..4144)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5060..5365
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     5244..5263
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5406..5530)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(5541..5891,5892..5894,5895..6503)
                     /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"
     CDS             6534..6557
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(6598..6615)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6604..6828
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6961..6980)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    7112..7159
                     /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             join(7167..7362,1..827)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
ORIGIN
        1 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggagttc agcgagagcc
       61 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg
      121 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc
      181 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat
      241 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg
      301 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg
      361 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg
      421 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat
      481 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc
      541 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc
      601 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag
      661 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta
      721 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg
      781 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag
      841 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
      901 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
      961 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     1021 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     1081 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     1141 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     1201 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1261 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1321 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1381 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1441 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1501 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1561 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1621 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1681 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1741 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1801 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1861 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     1921 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     1981 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2041 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2101 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2161 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2221 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2281 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2341 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2401 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2461 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2521 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2581 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2641 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2701 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     2761 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     2821 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     2881 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     2941 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3001 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3061 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3121 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3181 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3241 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     3301 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3361 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     3421 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     3481 tgcgctgctt cgcgatgtac gggccagata tacgcgtctc gagggatcca agctggccgc
     3541 acgtctccct atcagtgata gagaagtcga cacgtctcga gctccctatc agtgatagag
     3601 aaggtacgtc tagaacgtct ccctatcagt gatagagaag tcgacacgtc tcgagctccc
     3661 tatcagtgat agagaaggta ccccctatat aagcagagct cgtttagtga accgtcagat
     3721 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
     3781 cctccgcgga ggctggatcc tctagcgttt aaacttaagc tttagacaag tttgtacaaa
     3841 aaagctgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     3901 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat
     3961 taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt
     4021 aggatccgtc gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat
     4081 ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag
     4141 ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg
     4201 taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga
     4261 atgctcatcc ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg
     4321 ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg
     4381 aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg
     4441 gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca
     4501 atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg
     4561 cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg
     4621 cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat
     4681 tacaacagta ctgcgatgag tggcagggcg gggcgtaaag atctggatcc ggcttactaa
     4741 aagccagata acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg
     4801 atatgtatac ccgaagtatg tcaaaaagag gtatgctatg aagcagcgta ttacagtgac
     4861 agttgacagc gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg
     4921 gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa
     4981 aatcaggaag ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac
     5041 gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta
     5101 tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat
     5161 ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt
     5221 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
     5281 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
     5341 taacctgatg ttctggggaa tataaatgtc aggctccctt atacacagcc agtctgcagg
     5401 tcgaccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca
     5461 aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca gctttcttgt
     5521 acaaagtggt tgattctaga atgaaggaca acaccgtgcc cctgaagctg atcgccctgc
     5581 tggccaacgg cgagttccac agcggcgagc agctgggcga gaccctgggc atgagccggg
     5641 ccgccatcaa caagcacatc cagaccctgc gggactgggg cgtggacgtg ttcaccgtgc
     5701 ccggcaaggg ctacagcctg cccgagccca tccagctgct gaacgccaag cagatcctgg
     5761 gccagctgga cggcggcagc gtggccgtgc tgcccgtgat cgacagcacc aaccagtacc
     5821 tgctggaccg gatcggcgag ctgaagagcg gcgacgcctg catcgccgag taccagcagg
     5881 ccggccgggg cggccggggc cggaagtggt tcagcccctt cggcgccaac ctgtacctga
     5941 gcatgttctg gcggctggag cagggccccg ccgccgccat cggcctgagc ctggtgatcg
     6001 gcatcgtgat ggccgaggtg ctgcggaagc tgggcgccga caaggtgcgg gtgaagtggc
     6061 ccaacgacct gtacctgcag gaccggaagc tggccggcat cctggtggag ctgaccggca
     6121 agaccggcga cgccgcccag atcgtgatcg gcgccggcat caacatggcc atgcggcggg
     6181 tggaggagag cgtggtgaac cagggctgga tcaccctgca ggaggccggc atcaacctgg
     6241 accggaacac cctggccgcc atgctgatcc gggagctgcg ggccgccctg gagctgttcg
     6301 agcaggaggg cctggccccc tacctgagcc ggtgggagaa gctggacaac ttcatcaacc
     6361 ggcccgtgaa gctgatcatc ggcgacaagg agatcttcgg catcagccgg ggcatcgaca
     6421 agcagggcgc cctgctgctg gagcaggacg gcatcatcaa gccctggatg ggcggcgaga
     6481 tcagcctgcg gagcgccgag aaggggggat ctggccccgg cggcggcgcg ccagactaca
     6541 aagacgatga cgataaataa ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     6601 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     6661 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     6721 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     6781 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     6841 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     6901 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     6961 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     7021 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     7081 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     7141 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     7201 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     7261 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     7321 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ct
//
