LOCUS       sequence-421026-        5475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LR vector for integration of GFP into N2a FRT rtTA3 expression 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5475)
  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 5475)
  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 GFP minimal CMV pCDNA5
FEATURES             Location/Qualifiers
     source          1..5475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    14..135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(51..70)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     105..124
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(184..200)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(184..200)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(197..219)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    208..224
                     /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(232..238,239..256,257..262))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    277..298
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(415..432)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(586..1174)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(666..685)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1345..2136,2137..2205))
                     /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     1968..1987
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2206..2310)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2385..2404)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    2603..2621
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2640..2658
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2677..2695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2714..2732
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2754..2778
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2930..2953
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    2954..2978
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2981..2983,2984..2986,2987..3700)
                     /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             join(2981..2983,2984..2986,2987..3697)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3026..3047)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3287..3306)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3634..3655
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3702..3726)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3768..3785)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3774..3998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4131..4150)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4282..4329
                     /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             4337..5359
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
ORIGIN
        1 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
       61 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
      121 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
      181 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
      241 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
      301 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
      361 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
      421 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
      481 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
      541 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
      601 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
      661 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
      721 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
      781 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
      841 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
      901 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
      961 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     1021 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     1081 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     1141 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     1201 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     1261 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     1321 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     1381 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     1441 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     1501 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     1561 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     1621 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     1681 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     1741 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     1801 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     1861 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     1921 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     1981 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2041 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     2101 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     2161 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     2221 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2281 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     2341 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     2401 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
     2461 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
     2521 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgtctcgag
     2581 ggatccaagc tggccgcacg tctccctatc agtgatagag aagtcgacac gtctcgagct
     2641 ccctatcagt gatagagaag gtacgtctag aacgtctccc tatcagtgat agagaagtcg
     2701 acacgtctcg agctccctat cagtgataga gaaggtaccc cctatataag cagagctcgt
     2761 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
     2821 caccgggacc gatccagcct ccgcggaggc tggatcctct agcgtttaaa cttaagcttc
     2881 caccatggac tacaaagacc atgacggtga tacatatgat catgacatcg attacaagga
     2941 tgacgatgac aagacaagtt tgtacaaaaa agttggcacc atggtgagca agggcgagga
     3001 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     3061 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     3121 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     3181 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     3241 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     3301 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3361 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3421 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3481 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3541 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     3601 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3661 cgccgggatc actctcggca tggacgagct gtacaagtag tacccaactt tcttgtacaa
     3721 agtggtctga ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3781 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3841 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3901 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3961 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     4021 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4081 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4141 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4201 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4261 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     4321 aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     4381 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     4441 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     4501 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     4561 agtgcttgac attggggagt tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     4621 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     4681 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     4741 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     4801 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     4861 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     4921 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     4981 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     5041 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     5101 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     5161 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     5221 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     5281 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     5341 tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga
     5401 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5461 tctcatgctg gagtt
//
