LOCUS       sequence-318348-        5526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of 3xFLAG-NLS-DsDed-intN under the ZF6x6-C 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5526)
  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 5526)
  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: pJM577 ZF6x6-C 3xFLAG-NLS-DsDed-intN in TUPV2
FEATURES             Location/Qualifiers
     source          1..5526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      349..358
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             358..423
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             430..450
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             493..1167
                     /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(673..693)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1085..1108
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1569..1586)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1575..1799
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2568..2996
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2655..2674)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2865..2886
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3019..3215
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3066..3201
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3128..3147
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3335..3456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3372..3391)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3426..3445
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3505..3521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3505..3521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3518..3540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3529..3545
                     /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(3553..3559,3560..3577,3578..3583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3598..3619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3736..3753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3907..4495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3987..4006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4666..5457,5458..5526))
                     /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     5289..5308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtcgacg gatcgggaga tcgaagacgg gcaaaaccga tgttgacgga
      181 ctaatcctga caagatctgc gaggtgtagg ggacgcgagt gtaggggtga gctgtgtagg
      241 gggactaagt gtaggggacg cgagtgtagg ggtgagctgt gtaggggaac gctctctaga
      301 gggtatataa tgggggccac tagtctacta ccagagctca tcgctagcgc caccatggat
      361 tataaggatc acgatggcga ctacaaggac cacgatattg actacaaaga cgatgacgat
      421 aaaatggctc ctaagaaaaa gcgcaaagtc ggtatccatg gcgttcccgg atcaggctcc
      481 ggaagtggct ccatggatag cactgagaac gtcatcaagc ccttcatgcg cttcaaggtg
      541 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggcaag
      601 ccctacgagg gcacccagac cgccaagctc caggtgacca agggcggccc cctgcccttc
      661 gcctgggaca tcctgtcccc ccagttccag tacggctcca aggtgtacgt gaagcacccc
      721 gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg ggagaaggtg
      781 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct ccaggacggc
      841 accttcatct accacgtgaa gttcatcggc gtgaacttcc cctccgacgg ccccgtaatg
      901 cagaagaaaa ctctgggctg ggagccctcc accgagcgcc tgtacccccg cgacggcgtg
      961 ctgaagggcg agatccacaa ggcgctgaag ctgaagggcg gcggccacta cctggtggag
     1021 ttcaagtcaa tctacatggc caagaagccc gtgaagctgc ccggctacta ctacgtggac
     1081 tccaagctgg acatcacctc ccacaacgag gactacaccg tggtggagca gtacgagcgc
     1141 gccgaggccc gccaccacct gttccagtct ggttctggtt ctggttcagg atccggtggt
     1201 ggcgggtctg gaggaggagg tactggtgga ggtggcactg gtaccagaag cggatactgc
     1261 ctggacctga agacccaggt gcagacccct cagggcatga aggagatcag caacatccag
     1321 gtgggcgacc tggtgctgag caacaccggc tacaacgagg tgctgaacgt gttccccaag
     1381 agcaagaaga agagctacaa gatcaccctg gaggacggca aggagatcat ctgcagcgag
     1441 gagcacctgt tccccaccca gaccggcgag atgaacatca gcggcggcct gaaggagggc
     1501 atgtgcctgt acgtgaagga gtgagcggcc gctcgagtct agagggcccg tttaaacccg
     1561 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     1621 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     1681 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     1741 caagggggag gattgggaag tcaatagcag gcatgctggg gatgcggtgg gctctatggc
     1801 ttctgaggca ggtcgaagaa ctatttcctg gctgttacgc gagcgcatcg cgataagctc
     1861 tagagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
     1921 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
     1981 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
     2041 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt
     2101 aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgcgtac ggatggggct
     2161 ctagctctag agcttgatat cgaattcctg cagccccgct agagggacag ccccccccca
     2221 aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg
     2281 ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc
     2341 ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg
     2401 agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgaaaga gagatttaga
     2461 atgacagaat catagaacgg cctgggcgcg cctggtggcg aacgatcaac agacgggaga
     2521 ggactaaagt cttcgctgac agcgaccgga ctctaggggg tatccccacg cgccctgtag
     2581 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2641 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2701 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2761 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2821 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2881 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2941 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3001 ctgtggaatg tgtgtcagtt aggctcaatt agtcagcaac catagtcccg cccctaactc
     3061 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3121 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3181 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3241 ttttcggagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3301 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3361 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3421 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3481 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3541 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3601 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3661 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3721 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3781 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3841 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3901 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3961 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4021 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4081 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4141 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4201 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4261 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4321 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4381 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4441 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4501 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4561 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4621 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4681 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4741 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4801 accgcgagat ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4861 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4921 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4981 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5041 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5101 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5161 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5221 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5281 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5341 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5401 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5461 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5521 actcat
//
