LOCUS       sequence-421251-        6411 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 6411)
  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 6411)
  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'-3xFLAG minimal CMV pCDNA5
FEATURES             Location/Qualifiers
     source          1..6411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    482..603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(519..538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     573..592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(652..668)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(652..668)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(665..687)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    676..692
                     /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(700..706,707..724,725..730))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    745..766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(883..900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1054..1642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1134..1153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1813..2604,2605..2673))
                     /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     2436..2455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2674..2778)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2853..2872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    3071..3089
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3108..3126
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3145..3163
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3182..3200
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3222..3246
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3351..3475
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3500..3505,3506..3523,3524..3530)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3584..4243
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3650..3669)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4585..4890
                     /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     4769..4788
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4931..5055)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5066..5131
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(5172..5189)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5178..5402
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5535..5554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5686..5733
                     /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(5741..6411,1..352)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
ORIGIN
        1 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
       61 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
      121 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
      181 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
      241 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
      301 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact
      361 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
      421 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
      481 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
      541 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
      601 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
      661 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
      721 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
      781 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      841 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
      901 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
      961 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1021 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1081 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1141 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1201 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1261 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1321 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1381 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1441 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1501 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1561 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1621 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1681 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1741 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1801 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     1861 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     1921 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     1981 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2041 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2101 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2161 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2221 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2281 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2341 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2401 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2461 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     2521 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     2581 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     2641 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2701 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2761 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     2821 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2881 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     2941 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     3001 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtctcgaggg atccaagctg
     3061 gccgcacgtc tccctatcag tgatagagaa gtcgacacgt ctcgagctcc ctatcagtga
     3121 tagagaaggt acgtctagaa cgtctcccta tcagtgatag agaagtcgac acgtctcgag
     3181 ctccctatca gtgatagaga aggtaccccc tatataagca gagctcgttt agtgaaccgt
     3241 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
     3301 tccagcctcc gcggaggctg gatcctctag cgtttaaact taagctttag acaagtttgt
     3361 acaaaaaagc tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt
     3421 tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca ctatggcggc
     3481 cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt
     3541 gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac
     3601 tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca
     3661 gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa
     3721 gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct
     3781 gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga
     3841 tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg
     3901 gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg
     3961 ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc
     4021 agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt
     4081 cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc
     4141 gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa
     4201 tgaattacaa cagtactgcg atgagtggca gggcggggcg taaagatctg gatccggctt
     4261 actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata
     4321 tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca
     4381 gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg
     4441 gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag
     4501 cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg
     4561 ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc
     4621 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg
     4681 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
     4741 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
     4801 gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
     4861 gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct
     4921 gcaggtcgac catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt
     4981 atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc gttcagcttt
     5041 cttgtacaaa gtggttgatt ctagagacta caaagaccat gacggtgatt ataaagatca
     5101 tgacatcgat tacaaggatg acgatgacaa gtaactcgag tctagagggc ccgtttaaac
     5161 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     5221 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     5281 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5341 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     5401 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     5461 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5521 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5581 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5641 cctcgacccc aaaaaacttg attagggtga tggttcacgt acctagaagt tcctattccg
     5701 aagttcctat tctctagaaa gtataggaac ttccttggcc aaaaagcctg aactcaccgc
     5761 gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
     5821 ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct
     5881 gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc
     5941 atcggccgcg ctcccgattc cggaagtgct tgacattggg gagttcagcg agagcctgac
     6001 ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact
     6061 gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag
     6121 ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg
     6181 tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga
     6241 caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg
     6301 ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa
     6361 tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt c
//
