LOCUS       sequence-318792-        7186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-DsDed-intC-ZF10-NLS-HA under 
            the EF1-alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7186)
  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 7186)
  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: pJM529 EF1-alpha 3xFLAG-NLS-DsDed-intC-ZF10-NLS-HA 
            ...
FEATURES             Location/Qualifiers
     source          1..7186
                     /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             1642..1707
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1714..1734
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1777..2451
                     /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(1957..1977)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2369..2392
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2986..3006
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3019..3045
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3093..3110)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3099..3323
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4092..4520
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4179..4198)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4389..4410
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4543..4739
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4590..4725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4652..4671
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4859..4980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4896..4915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4950..4969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5029..5045)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5029..5045)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5042..5064)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5053..5069
                     /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(5077..5083,5084..5101,5102..5107))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5122..5143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5260..5277)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5431..6019)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5511..5530)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6190..6981,6982..7050))
                     /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     6813..6832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7051..7155)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcgaag acggcagaaa ggtatggcac gcctaatctg gacacaagat
       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 gcgggcgggt 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 ggattataag gatcacgatg gcgactacaa ggaccacgat
     1681 attgactaca aagacgatga cgataaaatg gctcctaaga aaaagcgcaa agtcggtatc
     1741 catggcgttc ccggatcagg ctccggaagt ggctccatgg atagcactga gaacgtcatc
     1801 aagcccttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     1861 atcgagggcg agggcgaggg caagccctac gagggcaccc agaccgccaa gctccaggtg
     1921 accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt ccagtacggc
     1981 tccaaggtgt acgtgaagca ccccgccgac atccccgact acaagaagct gtccttcccc
     2041 gagggcttca agtgggagaa ggtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     2101 caggactcct ccctccagga cggcaccttc atctaccacg tgaagttcat cggcgtgaac
     2161 ttcccctccg acggccccgt aatgcagaag aaaactctgg gctgggagcc ctccaccgag
     2221 cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc acaaggcgct gaagctgaag
     2281 ggcggcggcc actacctggt ggagttcaag tcaatctaca tggccaagaa gcccgtgaag
     2341 ctgcccggct actactacgt ggactccaag ctggacatca cctcccacaa cgaggactac
     2401 accgtggtgg agcagtacga gcgcgccgag gcccgccacc acctgttcca gtctggttct
     2461 ggttctggtt caggatccgg tggtggcggg tctggtggag gaggtagtgg tggaggtggc
     2521 tctgccacca tgatgctgaa gaagatcctg aagatcgagg agctggacga gagagagctg
     2581 atcgacatcg aggtgagcgg caaccacctg ttctacgcca acgacatcct gacccacaac
     2641 agcaacaggg gtggtggcgg gtctggtgga ggaggtagtg gtggaggtgg ctctggtacc
     2701 ggtggagcta gacccgggga gcgccccttc cagtgtcgca tttgcatgcg gaacttttcg
     2761 actaatcaaa aattggaagt tcatacccgt actcataccg gtgaaaaacc gtttcagtgt
     2821 cggatctgta tgcgaaattt ctccgttaga cataatttgc aaagacatct acgtacgcac
     2881 accggcgaga agccattcca atgccgaata tgcatgcgca acttcagtca acatccaaat
     2941 ttgactagac acctaaaaac ccacctgaga ggatctggct ctggccctaa gaagaagcgc
     3001 aaagttggct ctggcatgta cccatacgac gtaccagatt acgcttaggc ggccgctcga
     3061 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3121 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3181 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3241 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagtcaata gcaggcatgc
     3301 tggggatgcg gtgggctcta tggcttctga ggcaggtcga tgcaccgttc atatgttatc
     3361 gggacagcgc atcgcgataa gctctagagg gacagccccc ccccaaagcc cccagggatg
     3421 taattacgtc cctcccccgc tagggggcag cagcgagccg cccggggctc cgctccggtc
     3481 cggcgctccc cccgcatccc cgagccggca gcgtgcgggg acagcccggg cacggggaag
     3541 gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct
     3601 ggggggatac ggggaaaaag ctttaggctg aaagagagat ttagaatgac agaatcatag
     3661 aacggcctgc gtacggatgg ggctctagct ctagagcttg atatcgaatt cctgcagccc
     3721 cgctagaggg acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct
     3781 agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc
     3841 gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct
     3901 ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaagc
     3961 tttaggctga aagagagatt tagaatgaca gaatcataga acggcctggg cgcgcctggt
     4021 ggcgaacgat caacagacgg gagaggtgtg aagtcttcgc tgacagcgac cggactctag
     4081 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4141 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4201 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4261 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4321 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4381 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4441 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4501 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttaggctc aattagtcag
     4561 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4621 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     4681 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     4741 agctcccggg agcttgtata tccattttcg gaggttgggc ttcggaatcg ttttccggga
     4801 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4861 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4921 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4981 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5041 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5101 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5161 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5221 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5281 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5341 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5401 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5461 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5521 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5581 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5641 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5701 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5761 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5821 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5881 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5941 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6001 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6061 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6121 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6181 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6241 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6301 atctggcccc agtgctgcaa tgataccgcg agatccacgc tcaccggctc cagatttatc
     6361 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6421 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6481 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6541 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6601 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6661 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6721 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6781 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6841 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6901 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6961 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7021 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7081 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7141 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
