LOCUS       sequence-421250-        6407 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 6407)
  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 6407)
  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'-3xFLAG minimal CMV pCDNA5
FEATURES             Location/Qualifiers
     source          1..6407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(541..588)
                     /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     720..739
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    872..1096
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1085..1102
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     protein_bind    1144..1268
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1309..1614)
                     /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
                     VSRELYPVVHIGDESWRMLTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             complement(1956..2615)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2530..2549
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2669..2675,2676..2693,2694..2699))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2724..2848)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2849..2872)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3024..3048)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3070..3088
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3107..3125
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3144..3162
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3181..3199
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3398..3417
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3492..3596
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3597..3665,3666..4457)
                     /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(3815..3834)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4628..5216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5117..5136
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5370..5387
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5504..5525
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5540..5545,5546..5563,5564..5570)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5578..5594
                     /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     5583..5605
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5602..5618
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5602..5618
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5667..5788
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5678..5697)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5732..5751
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5918..6407,1..533))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
ORIGIN
        1 tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac
       61 acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct
      121 tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatcgcatcc
      181 atggcctccg cgaccggctg cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac
      241 gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa ctccccaatg
      301 tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct
      361 ttgtagaaac catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg
      421 aagctgaaag cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg
      481 aacttttcga tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttggccaag
      541 gaagttccta tactttctag agaataggaa cttcggaata ggaacttcta ggtacgtgaa
      601 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      661 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      721 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      781 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtgggg atacccccta
      841 gagccccagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc cccagcatgc
      901 ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata
      961 gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg
     1021 ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg
     1081 caactagaag gcacagtcga ggctgatcag cgggtttaaa cgggccctct agactcgagt
     1141 cagaccactt tgtacaagaa agctgaacga gaaacgtaaa atgatataaa tatcaatata
     1201 ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag
     1261 tcactatggt cgacctgcag actggctgtg tataagggag cctgacattt atattcccca
     1321 gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg tggctgagat cagccacttc
     1381 ttccccgata acggagaccg gcacactggc catatcggtg gtcaacatgc gccagctttc
     1441 atccccgata tgcaccaccg ggtaaagttc acgggagact ttatctgaca gcagacgtgc
     1501 actggccagg gggatcacca tccgtcgccc gggcgtgtca ataatatcac tctgtacatc
     1561 cacaaacaga cgataacggc tctctctttt ataggtgtaa accttaaact gcatttcacc
     1621 agcccctgtt ctcgtcagca aaagagccgt tcatttcaat aaaccgggcg acctcagcca
     1681 tcccttcctg attttccgct ttccagcgtt cggcacgcag acgacgggct tcattctgca
     1741 tggttgtgct taccagaccg gagatattga catcatatat gccttgagca actgatagct
     1801 gtcgctgtca actgtcactg taatacgctg cttcatagca tacctctttt tgacatactt
     1861 cgggtataca tatcagtata tattcttata ccgcaaaaat cagcgcgcaa atacgcatac
     1921 tgttatctgg cttttagtaa gccggatcca gatctttacg ccccgccctg ccactcatcg
     1981 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
     2041 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     2101 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
     2161 ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
     2221 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     2281 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     2341 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat
     2401 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     2461 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     2521 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     2581 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
     2641 ctcgacggat cctaactcaa aatccacaca ttatacgagc cggaagcata aagtgtaaag
     2701 cctggggtgc ctaatgcggc cgccatagtg actggatatg ttgtgtttta cagtattatg
     2761 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
     2821 ctcgttcagc ttttttgtac aaacttgtct tgtcatcgtc atccttgtaa tcgatgtcat
     2881 gatcatatgt atcaccgtca tggtctttgt agtccatggt ggaagcttaa gtttaaacgc
     2941 tagaggatcc agcctccgcg gaggctggat cggtcccggt gtcttctatg gaggtcaaaa
     3001 cagcgtggat ggcgtctcca ggcgatctga cggttcacta aacgagctct gcttatatag
     3061 ggggtacctt ctctatcact gatagggagc tcgagacgtg tcgacttctc tatcactgat
     3121 agggagacgt tctagacgta ccttctctat cactgatagg gagctcgaga cgtgtcgact
     3181 tctctatcac tgatagggag acgtgcggcc agcttggatc cctcgagacg cgtatatctg
     3241 gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg
     3301 tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc
     3361 aacacacaag cagggagcag atactggctt aactatgcgg catcagagca gattgtactg
     3421 agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt ggcacttttc
     3481 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3541 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3601 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3661 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3721 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3781 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3841 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3901 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3961 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4021 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4081 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4141 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4201 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4261 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4321 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4381 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4441 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4501 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4561 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4621 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4681 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4741 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4801 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4861 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4921 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4981 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5041 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5101 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5161 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5221 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5281 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5341 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     5401 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5461 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     5521 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     5581 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctagc
     5641 tagaggtcga cggtatacag acatgataag atacattgat gagtttggac aaaccacaac
     5701 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     5761 aaccattata agctgcaata aacaagttgg ggtgggcgaa gaactccagc atgagatccc
     5821 cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat
     5881 agaaggcggc ggtggaatcg aaatctcgta gtacgtgcta ttcctttgcc ctcggacgag
     5941 tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg
     6001 ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg
     6061 cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat
     6121 agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat cctgcaagct
     6181 ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc
     6241 agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa
     6301 tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca
     6361 cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctca
//
