LOCUS       sequence-325010-        6988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mMoClo TUPV8, with EF1alpha promoter and EBFP2-P2A-BlastR in the 
            MCS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6988)
  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 6988)
  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: pJM457 EF1alpha EBFP2-P2A-BlastR (TUPV8)
FEATURES             Location/Qualifiers
     source          1..6988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        350..1532
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          581..1523
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1480..1500
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1534..1558
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1633..1642
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1639..1641,1642..1644,1645..2355)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1684..1705)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             2383..2439
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2452..2850
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2895..2912)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2901..3125
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3894..4322
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3981..4000)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4191..4212
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4345..4541
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4392..4527
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4454..4473
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4661..4782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4698..4717)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4752..4771
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4831..4847)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4831..4847)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4844..4866)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4855..4871
                     /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(4879..4885,4886..4903,4904..4909))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4924..4945
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5062..5079)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5233..5821)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5313..5332)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5992..6783,6784..6852))
                     /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     6615..6634
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6853..6957)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcgaag acggaacgaa gccaccttaa cacgcgatga tattgaagat
       61 ctggggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
      121 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
      181 gggccagata tacgcgtgag taattcatac aaaaggactc gcccctgcct tggggaatcc
      241 cagggaccgt cgttaaactc ccactaacgt agaacccaga gatcgctgcg ttcccgcccc
      301 ctcacccgcc cgctctcgtc atcactgagg tggagaagag catgcgtgag gctccggtgc
      361 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
      421 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
      481 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
      541 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
      601 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacgcc
      661 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
      721 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcttgggc
      781 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
      841 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata
      901 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
      961 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     1021 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     1081 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     1141 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc
     1201 gctcgggaga tcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     1261 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     1321 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     1381 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     1441 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     1501 aaagtttttt tcttccattt caggtgtcgt gagagctcgt ttagtgaacc gtcagatcgc
     1561 ctggagacgc catccacgct gttttgacct ccatagaaga gaacgggacc gatccaaatt
     1621 ctcgaggcta gcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1681 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgag gggcgagggc
     1741 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1801 cccgtgccct ggcccaccct cgtgaccacc ctgagccacg gcgtgcagtg cttcgcccgc
     1861 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1921 caggagcgca ccatcttctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag
     1981 ttcgagggcg acaccctagt gaaccgcatc gagctgaagg gcgtcgactt caaggaggac
     2041 ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccacaacat ctatatcatg
     2101 gccgtcaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa cgtggaggac
     2161 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2221 ctgctgcccg acagccacta cctgagcacc cagtccgtgc tgagcaaaga ccccaacgag
     2281 aagcgcgatc acatggtcct gctggagttc cgcaccgccg ccgggatcac tctcggcatg
     2341 gacgagctgt acaaggaatt cggtagcaag cttggtagtg gagcaactaa cttttccctg
     2401 ctcaaacaag ccggggatgt agaggagaac cccggccccg ccccccttaa gatggccaag
     2461 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     2521 cccatctctg aggactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     2581 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     2641 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     2701 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     2761 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     2821 ctgccctctg gttatgtgtg ggagggctaa gcggccgctc gagtctagag ggcccgttta
     2881 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2941 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3001 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3061 ggacagcaag ggggaggatt gggaagtcaa tagcaggcat gctggggatg cggtgggctc
     3121 tatggcttct gaggcaggtc gaatgagctg tctcgaaccg aaggcacagc gcatcgcgat
     3181 aagctctaga gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc
     3241 gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc
     3301 cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt
     3361 cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa
     3421 agctttaggc tgaaagagag atttagaatg acagaatcat agaacggcct gcgtacggat
     3481 ggggctctag ctctagagct tgatatcgaa ttcctgcagc cccgctagag ggacagcccc
     3541 cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc
     3601 gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg
     3661 gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc
     3721 tctttgagcc tgcagacacc tggggggata cggggaaaaa gctttaggct gaaagagaga
     3781 tttagaatga cagaatcata gaacggcctg ggcgcgcctg gtggcgaacg atcaacagac
     3841 gggagaggct tcaagtcttc gctgacagcg accggactct agggggtatc cccacgcgcc
     3901 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3961 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4021 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4081 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4141 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4201 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4261 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4321 ttaattctgt ggaatgtgtg tcagttaggc tcaattagtc agcaaccata gtcccgcccc
     4381 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4441 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     4501 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     4561 tatccatttt cggaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4621 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     4681 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4741 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4801 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4861 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4921 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4981 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5041 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5101 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5161 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5221 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5281 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5341 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5401 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5461 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5521 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5581 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5641 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5701 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5761 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5821 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5881 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5941 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6001 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6061 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6121 aatgataccg cgagatccac gctcaccggc tccagattta tcagcaataa accagccagc
     6181 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6241 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6301 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6361 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6421 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6481 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6541 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6601 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6661 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6721 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6781 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6841 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6901 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6961 atttccccga aaagtgccac ctgacgtc
//
