LOCUS       sequence-318354-        6418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mMoClo TUPV, with EF1alpha promoter and VP16-ZFa in the MCS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6418)
  AUTHORS   Donahue PS, Draut JW, Muldoon JJ, Edelstein HI, Bagheri N, Leonard 
            JN
  TITLE     The COMET toolkit for composing customizable genetic programs in 
            mammalian cells.
  JOURNAL   Nat Commun. 2020 Feb 7;11(1):779. doi: 10.1038/s41467-019-14147-5.
  PUBMED    32034124
REFERENCE   2  (bases 1 to 6418)
  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"; journalName: "Nat 
            Commun."; date: "2020-02-7"; volume: "11(1):779. doi"; pages: " 
            10.1038/s41467-019-14147-5"
COMMENT     Sequence Label: pJM466 EF1a VP16-ZF43 (TUPV2)
FEATURES             Location/Qualifiers
     source          1..6418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          231..1173
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1130..1150
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1184..1208
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1283..1292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1292..1357
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1364..1384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1975..1992)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1981..2205
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2974..3402
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3061..3080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3271..3292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3425..3621
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3472..3607
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3534..3553
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3741..3862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3778..3797)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3832..3851
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3911..3927)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3911..3927)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3924..3946)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3935..3951
                     /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(3959..3965,3966..3983,3984..3989))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4004..4025
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4142..4159)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4313..4901)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4393..4412)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5072..5863,5864..5932))
                     /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     5695..5714
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5933..6037)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6418,1..1182)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
ORIGIN
        1 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
       61 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
      121 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
      181 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
      241 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
      301 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
      361 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
      421 tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
      481 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
      541 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
      601 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
      661 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
      721 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
      781 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
      841 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
      901 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
      961 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     1021 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     1081 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     1141 acagtggttc aaagtttttt tcttccattt caggtgtcgt gagagctcgt ttagtgaacc
     1201 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga gaccgggacc
     1261 gatccaaatt ctcgaggcta gcgccaccat ggattataag gatcacgatg gcgactacaa
     1321 ggaccacgat attgactaca aagacgatga cgataaaatg gctcctaaga aaaagcgcaa
     1381 agtcggtatc catggcgttc ccgtcgacgg tggactgcag gccccaccaa ccgatgtttc
     1441 tttaggtgat gagttacatt tggacggtga ggacgtagct atggcacatg cagatgcctt
     1501 agacgatttt gacttggata tgttaggaga tggagattca ccaggacctg gtttcacacc
     1561 acacgattca gctccatacg gtgctttgga catggccgat ttcgaatttg aacagatgtt
     1621 tactgatgcc ttgggtatag acgaatacgg tggtggagct agacccgggg agcgcccctt
     1681 ccagtgtcgc atttgcatgc ggaacttttc gcgccaggac aggcttgaca ggcatacccg
     1741 tactcatacc ggtgaaaaac cgtttcagtg tcggatctgt atgcgaaatt tctcccagaa
     1801 ggagcacttg gcggggcatc tacgtacgca caccggcgag aagccattcc aatgccgaat
     1861 atgcatgcgc aacttcagtc gccgcgacaa cctgaaccgg cacctaaaaa cccacctgag
     1921 aggatcttaa gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga
     1981 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2041 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2101 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     2161 gggaagtcaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcaggtc
     2221 gaagaactat ttcctggctg ttacgcgagc gcatcgcgat aagctctaga gggacagccc
     2281 ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc
     2341 cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg
     2401 ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg
     2461 ctctttgagc ctgcagacac ctggggggat acggggaaaa agctttaggc tgaaagagag
     2521 atttagaatg acagaatcat agaacggcct gcgtacggat ggggctctag ctctagagct
     2581 tgatatcgaa ttcctgcagc cccgctagag ggacagcccc cccccaaagc ccccagggat
     2641 gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt
     2701 ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa
     2761 ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc
     2821 tggggggata cggggaaaaa gctttaggct gaaagagaga tttagaatga cagaatcata
     2881 gaacggcctg ggcgcgcctg gtggcgaacg atcaacagac gggagaggac taaagtcttc
     2941 gctgacagcg accggactct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3001 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3061 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3121 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3181 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3241 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3301 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3361 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     3421 tcagttaggc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3481 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3541 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3601 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggaggttgg
     3661 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3721 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     3781 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3841 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     3901 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3961 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4021 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4081 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4141 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4201 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4261 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4321 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4381 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4441 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4501 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4561 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4621 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4681 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4741 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4801 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4861 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4921 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4981 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5041 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5101 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5161 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagatccac
     5221 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5281 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5341 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5401 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5461 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5521 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5581 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5641 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5701 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5761 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5821 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5881 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5941 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6001 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6061 ctgacgtcga cggatcggga gatcgaagac gggcaaaacc gatgttgacg gactaatcct
     6121 gacaagatct ggggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     6181 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     6241 cgatgtacgg gccagatata cgcgtgagta attcatacaa aaggactcgc ccctgccttg
     6301 gggaatccca gggaccgtcg ttaaactccc actaacgtag aacccagaga tcgctgcgtt
     6361 cccgccccct cacccgcccg ctctcgtcat cactgaggtg gagaagagca tgcgtgag
//
