LOCUS       sequence-317923-        7180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-DsDed-intC-ZF1-NLS-HA under 
            the EF1-alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7180)
  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 7180)
  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: pJM525 EF1-alpha 3xFLAG-NLS-DsDed-intC-ZF1-NLS-HA 
            i...
FEATURES             Location/Qualifiers
     source          1..7180
                     /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"
     CDS             2630..2650
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2663..2689
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2737..2754)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2743..2967
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3736..4164
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3823..3842)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4033..4054
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4187..4383
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4234..4369
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4296..4315
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4503..4624
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4540..4559)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4594..4613
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4673..4689)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4673..4689)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4686..4708)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4697..4713
                     /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(4721..4727,4728..4745,4746..4751))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4766..4787
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4904..4921)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5075..5663)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5155..5174)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5834..6625,6626..6694))
                     /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     6457..6476
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6695..6799)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(7180,1..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 tctggtggag gaggtagtgg
     2161 tggaggtggc tctgccacca tgatgctgaa gaagatcctg aagatcgagg agctggacga
     2221 gagagagctg atcgacatcg aggtgagcgg caaccacctg ttctacgcca acgacatcct
     2281 gacccacaac agcaacaggg gtggtggcgg gtctggtgga ggaggtagtg gtggaggtgg
     2341 ctctggtacc gctagacccg gggagcgccc cttccagtgt cgcatttgca tgcggaactt
     2401 ttcgcgccag gacaggcttg acaggcatac ccgtactcat accggtgaaa aaccgtttca
     2461 gtgtcggatc tgtatgcgaa atttctccca gaaggagcac ttggcggggc atctacgtac
     2521 gcacaccggc gagaagccat tccaatgccg aatatgcatg cgcaacttca gtcgccgcga
     2581 caacctgaac cggcacctaa aaacccacct gagagaattc ggctctggcc ctaagaagaa
     2641 gcgcaaagtt ggctctggca tgtacccata cgacgtacca gattacgctt aggcggccgc
     2701 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2761 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2821 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2881 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagtc aatagcaggc
     2941 atgctgggga tgcggtgggc tctatggctt ctgaggcagg tcgaagaact atttcctggc
     3001 tgttacgcga gcgcatcgcg ataagctcta gagggacagc ccccccccaa agcccccagg
     3061 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     3121 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     3181 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     3241 acctgggggg atacggggaa aaagctttag gctgaaagag agatttagaa tgacagaatc
     3301 atagaacggc ctgcgtacgg atggggctct agctctagag cttgatatcg aattcctgca
     3361 gccccgctag agggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc
     3421 cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat
     3481 ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt
     3541 tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa
     3601 aagctttagg ctgaaagaga gatttagaat gacagaatca tagaacggcc tgggcgcgcc
     3661 tggtggcgaa cgatcaacag acgggagagg actaaagtct tcgctgacag cgaccggact
     3721 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3781 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3841 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3901 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3961 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4021 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4081 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4141 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag gctcaattag
     4201 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4261 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4321 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4381 aaaaagctcc cgggagcttg tatatccatt ttcggaggtt gggcttcgga atcgttttcc
     4441 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4501 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4561 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4621 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4681 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4741 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4801 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4861 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4921 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4981 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5041 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5101 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5161 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5221 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5281 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5341 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5401 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5461 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5521 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5581 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5641 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5701 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5761 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5821 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5881 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5941 taccatctgg ccccagtgct gcaatgatac cgcgagatcc acgctcaccg gctccagatt
     6001 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6061 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6121 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6181 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6241 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6301 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6361 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6421 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6481 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6541 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6601 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6661 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6721 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6781 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     6841 gagatcgaag acgggcaaaa ccgatgttga cggactaatc ctgacaagat ctggggaggt
     6901 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     6961 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     7021 tacgcgtgag taattcatac aaaaggactc gcccctgcct tggggaatcc cagggaccgt
     7081 cgttaaactc ccactaacgt agaacccaga gatcgctgcg ttcccgcccc ctcacccgcc
     7141 cgctctcgtc atcactgagg tggagaagag catgcgtgag
//
