LOCUS       sequence-318350-        6946 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-DsDed-intN under the EF1-alpha
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6946)
  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 6946)
  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: pJM555 EF1-alpha 3xFLAG-NLS-DsDed-intN in TUPV2
FEATURES             Location/Qualifiers
     source          1..6946
                     /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"
     CDS             1427..2101
                     /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(1607..1627)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2019..2042
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2503..2520)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2509..2733
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3502..3930
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3589..3608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3799..3820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3953..4149
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4000..4135
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4062..4081
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4269..4390
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4306..4325)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4360..4379
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4439..4455)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4439..4455)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4452..4474)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4463..4479
                     /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(4487..4493,4494..4511,4512..4517))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4532..4553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4670..4687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4841..5429)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4921..4940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5600..6391,6392..6460))
                     /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     6223..6242
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6461..6565)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6946,1..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 gaacgggacc
     1261 gatccaaatt ctcgaggcta gcgccaccat ggattataag gatcacgatg gcgactacaa
     1321 ggaccacgat attgactaca aagacgatga cgataaaatg gctcctaaga aaaagcgcaa
     1381 agtcggtatc catggcgttc ccggatcagg ctccggaagt ggctccatgg atagcactga
     1441 gaacgtcatc aagcccttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1501 cgagttcgag atcgagggcg agggcgaggg caagccctac gagggcaccc agaccgccaa
     1561 gctccaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt
     1621 ccagtacggc tccaaggtgt acgtgaagca ccccgccgac atccccgact acaagaagct
     1681 gtccttcccc gagggcttca agtgggagaa ggtgatgaac ttcgaggacg gcggcgtggt
     1741 gaccgtgacc caggactcct ccctccagga cggcaccttc atctaccacg tgaagttcat
     1801 cggcgtgaac ttcccctccg acggccccgt aatgcagaag aaaactctgg gctgggagcc
     1861 ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc acaaggcgct
     1921 gaagctgaag ggcggcggcc actacctggt ggagttcaag tcaatctaca tggccaagaa
     1981 gcccgtgaag ctgcccggct actactacgt ggactccaag ctggacatca cctcccacaa
     2041 cgaggactac accgtggtgg agcagtacga gcgcgccgag gcccgccacc acctgttcca
     2101 gtctggttct ggttctggtt caggatccgg tggtggcggg tctggaggag gaggtactgg
     2161 tggaggtggc actggtacca gaagcggata ctgcctggac ctgaagaccc aggtgcagac
     2221 ccctcagggc atgaaggaga tcagcaacat ccaggtgggc gacctggtgc tgagcaacac
     2281 cggctacaac gaggtgctga acgtgttccc caagagcaag aagaagagct acaagatcac
     2341 cctggaggac ggcaaggaga tcatctgcag cgaggagcac ctgttcccca cccagaccgg
     2401 cgagatgaac atcagcggcg gcctgaagga gggcatgtgc ctgtacgtga aggagtgagc
     2461 ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     2521 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2581 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2641 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagtcaata
     2701 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcaggtcga agaactattt
     2761 cctggctgtt acgcgagcgc atcgcgataa gctctagagg gacagccccc ccccaaagcc
     2821 cccagggatg taattacgtc cctcccccgc tagggggcag cagcgagccg cccggggctc
     2881 cgctccggtc cggcgctccc cccgcatccc cgagccggca gcgtgcgggg acagcccggg
     2941 cacggggaag gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct
     3001 gcagacacct ggggggatac ggggaaaaag ctttaggctg aaagagagat ttagaatgac
     3061 agaatcatag aacggcctgc gtacggatgg ggctctagct ctagagcttg atatcgaatt
     3121 cctgcagccc cgctagaggg acagcccccc cccaaagccc ccagggatgt aattacgtcc
     3181 ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc
     3241 ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga
     3301 tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg
     3361 gggaaaaagc tttaggctga aagagagatt tagaatgaca gaatcataga acggcctggg
     3421 cgcgcctggt ggcgaacgat caacagacgg gagaggacta aagtcttcgc tgacagcgac
     3481 cggactctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3541 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3601 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3661 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3721 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3781 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3841 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3901 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttaggctc
     3961 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4021 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4081 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4141 ttttgcaaaa agctcccggg agcttgtata tccattttcg gaggttgggc ttcggaatcg
     4201 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4261 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4321 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4381 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4441 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4501 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4561 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4621 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4681 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4741 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4801 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4861 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4921 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4981 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5041 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5101 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5161 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5221 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5281 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5341 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5401 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5461 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5521 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5581 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5641 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5701 agggcttacc atctggcccc agtgctgcaa tgataccgcg agatccacgc tcaccggctc
     5761 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5821 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5881 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5941 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6001 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6061 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6121 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6181 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6241 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6301 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6361 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6421 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6481 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6541 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6601 gatcgggaga tcgaagacgg gcaaaaccga tgttgacgga ctaatcctga caagatctgg
     6661 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     6721 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     6781 cagatatacg cgtgagtaat tcatacaaaa ggactcgccc ctgccttggg gaatcccagg
     6841 gaccgtcgtt aaactcccac taacgtagaa cccagagatc gctgcgttcc cgccccctca
     6901 cccgcccgct ctcgtcatca ctgaggtgga gaagagcatg cgtgag
//
