LOCUS       sequence-317932-        7396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of TC 
            3xFLAG-FRB-FGFR4-PRS(M)-(GS)-NLS-DsDed-ZF1 under the EF1-alpha 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7396)
  AUTHORS   Muldoon JJ, Kandula V, Hong M, Donahue PS, Boucher JD, Bagheri N, 
            Leonard JN
  TITLE     Model-guided design of mammalian genetic programs.
  JOURNAL   Sci Adv. 2021 Feb 19;7(8). pii: 7/8/eabe9375. doi: 
            10.1126/sciadv.abe9375. Print 2021 Feb.
  PUBMED    33608279
REFERENCE   2  (bases 1 to 7396)
  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: "Sci Adv."; 
            date: "2021-02-19"; volume: "7(8). pii: 7/8/eabe9375. doi"; pages: "
            10.1126/sciadv.abe9375. Print 2021 Feb"
COMMENT     Sequence Label: pJM621 EF1-alpha TC
            3xFLAG-FRB-FGFR4-PRS(M)-(GS)-NL...
FEATURES             Location/Qualifiers
     source          1..7396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      599..1027
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(686..705)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     896..917
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1050..1246
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1097..1232
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1159..1178
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    1366..1487
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1403..1422)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1457..1476
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1536..1552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1536..1552)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1549..1571)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1560..1576
                     /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(1584..1590,1591..1608,1609..1614))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1629..1650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1767..1784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1938..2526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2018..2037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2697..3488,3489..3557))
                     /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     3320..3339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3558..3662)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4043..5225
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4274..5216
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5173..5193
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     5227..5251
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5410..5475
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5875..5895
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             5902..5922
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5965..6639
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWEKVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(6145..6165)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     6557..6580
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(6996..7013)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7002..7226
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc
       61 gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg gggatacggg
      121 gaaaaagctt taggctgaaa gagagattta gaatgacaga atcatagaac ggcctgcgta
      181 cggatggggc tctagctcta gagcttgata tcgaattcct gcagccccgc tagagggaca
      241 gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc
      301 gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt
      361 gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc
      421 gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt aggctgaaag
      481 agagatttag aatgacagaa tcatagaacg gcctgggcgc gcctggtggc gaacgatcaa
      541 cagacgggag aggttacaag tcttcgctga cagcgaccgg actctagggg gtatccccac
      601 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
      661 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
      721 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
      781 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
      841 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
      901 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
      961 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1021 gcgaattaat tctgtggaat gtgtgtcagt taggctcaat tagtcagcaa ccatagtccc
     1081 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     1141 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     1201 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     1261 ttgtatatcc attttcggag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     1321 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     1381 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     1441 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     1501 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     1561 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     1621 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1681 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1741 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1801 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1861 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1921 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1981 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2041 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2101 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2161 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2221 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2281 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2341 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2401 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2461 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2521 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2581 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2641 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2701 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2761 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2821 gctgcaatga taccgcgaga tccacgctca ccggctccag atttatcagc aataaaccag
     2881 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2941 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3001 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3061 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3121 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3181 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3241 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3301 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3361 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3421 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3481 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3541 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3601 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3661 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg aagacggact
     3721 aaatagtagt tcagacgccg ttaagcgcaa gatctgggga ggtcgctgag tagtgcgcga
     3781 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     3841 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt gagtaattca
     3901 tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa ctcccactaa
     3961 cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc gtcatcactg
     4021 aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc
     4081 cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg
     4141 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     4201 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     4261 cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta
     4321 tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg attcttgatc
     4381 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
     4441 cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg cgaatctggt
     4501 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
     4561 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc
     4621 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
     4681 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
     4741 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
     4801 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
     4861 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac
     4921 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     4981 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag
     5041 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
     5101 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat
     5161 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt
     5221 cgtgagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     5281 cctccataga agagaacggg accgatccaa attctcgagg ctagcgccat catgtgccga
     5341 gccatctctc ttaggcgctt gctgctgctg ctgctgcagc tgtcacaact cctagctgtc
     5401 actcaagggg attacaagga tcacgatggg gattacaaag atcatgacat tgactacaag
     5461 gatgacgatg ataagatgct cgagatgtgg catgaaggcc tggaagaggc atctcgtttg
     5521 tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc tggagccctt gcatgctatg
     5581 atggaacggg gcccccagac tctgaaggaa acatccttta atcaggccta tggtcgagat
     5641 ttaatggagg cccaagagtg gtgcaggaag tacatgaaat cagggaatgt caaggacctc
     5701 ctccaagcct gggacctcta ttatcatgtg ttccgacgaa tctcaaagac tagaggagga
     5761 tctggaggct ctgggggttc aggcggatca ggaggcagtg gcgggtcaac cggtattata
     5821 ctgtatgcta gtggcagtct ggcattggca gtcctcctgc tgctcgccgg gttggagaac
     5881 ctgtattttc agatgggatc tcctaagaaa aagcgcaaag tcggtatcca tggcgttccc
     5941 ggatcaggct ccggaagtgg ctccatggat agcactgaga acgtcatcaa gcccttcatg
     6001 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     6061 ggcgagggca agccctacga gggcacccag accgccaagc tccaggtgac caagggcggc
     6121 cccctgccct tcgcctggga catcctgtcc ccccagttcc agtacggctc caaggtgtac
     6181 gtgaagcacc ccgccgacat ccccgactac aagaagctgt ccttccccga gggcttcaag
     6241 tgggagaagg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     6301 ctccaggacg gcaccttcat ctaccacgtg aagttcatcg gcgtgaactt cccctccgac
     6361 ggccccgtaa tgcagaagaa aactctgggc tgggagccct ccaccgagcg cctgtacccc
     6421 cgcgacggcg tgctgaaggg cgagatccac aaggcgctga agctgaaggg cggcggccac
     6481 tacctggtgg agttcaagtc aatctacatg gccaagaagc ccgtgaagct gcccggctac
     6541 tactacgtgg actccaagct ggacatcacc tcccacaacg aggactacac cgtggtggag
     6601 cagtacgagc gcgccgaggc ccgccaccac ctgttccagt ctggttctgg ttctggttca
     6661 ggatccggta ccggtggagc tagacccggg gagcgcccct tccagtgtcg catttgcatg
     6721 cggaactttt cgcgccagga caggcttgac aggcataccc gtactcatac cggtgaaaaa
     6781 ccgtttcagt gtcggatctg tatgcgaaat ttctcccaga aggagcactt ggcggggcat
     6841 ctacgtacgc acaccggcga gaagccattc caatgccgaa tatgcatgcg caacttcagt
     6901 cgccgcgaca acctgaaccg gcacctaaaa acccacctga gaggatctta agcggccgct
     6961 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     7021 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     7081 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     7141 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagtca atagcaggca
     7201 tgctggggat gcggtgggct ctatggcttc tgaggcaggt cgaagagact gcccgacaca
     7261 tcttagtgag cgcatcgcga taagctctag agggacagcc cccccccaaa gcccccaggg
     7321 atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg
     7381 gtccggcgct cccccc
//
