LOCUS       sequence-318365-        4981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-VP16-intN under the CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4981)
  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 4981)
  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: pJB004 CMV 3xFLAG-NLS-VP16-intN
FEATURES             Location/Qualifiers
     source          1..4981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             676..741
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             748..768
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1377..1394)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1383..1607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1653..2081
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1740..1759)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1950..1971
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2104..2300
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2151..2286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2213..2232
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2420..2541
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2457..2476)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2511..2530
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2590..2606)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2590..2606)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2603..2625)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2614..2630
                     /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(2638..2644,2645..2662,2663..2668))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2683..2704
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2821..2838)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2992..3580)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3072..3091)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3751..4542,4543..4611))
                     /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     4374..4393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4612..4716)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4791..4810)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag tcccaagctg
      661 gctagcgcca ccatggatta taaggatcac gatggcgact acaaggacca cgatattgac
      721 tacaaagacg atgacgataa aatggctcct aagaaaaagc gcaaagtcgg tatccatggc
      781 gttcccggat ccgtcgacgg tggactgcag gccccaccaa ccgatgtttc tttaggtgat
      841 gagttacatt tggacggtga ggacgtagct atggcacatg cagatgcctt agacgatttt
      901 gacttggata tgttaggaga tggagattca ccaggacctg gtttcacacc acacgattca
      961 gctccatacg gtgctttgga catggccgat ttcgaatttg aacagatgtt tactgatgcc
     1021 ttgggtatag acgaatacgg tggtggaggt accagaagcg gatactgcct ggacctgaag
     1081 acccaggtgc agacccctca gggcatgaag gagatcagca acatccaggt gggcgacctg
     1141 gtgctgagca acaccggcta caacgaggtg ctgaacgtgt tccccaagag caagaagaag
     1201 agctacaaga tcaccctgga ggacggcaag gagatcatct gcagcgagga gcacctgttc
     1261 cccacccaga ccggcgagat gaacatcagc ggcggcctga aggagggcat gtgcctgtac
     1321 gtgaaggagt gagcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     1381 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1441 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1501 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1561 ttgggaagtc aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1621 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     1681 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1741 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1801 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1861 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1921 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1981 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2041 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2101 cagttaggct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2161 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2221 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2281 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggaggttggg
     2341 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     2401 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     2461 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2521 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     2581 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2641 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2701 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2761 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2821 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2881 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2941 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3001 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3061 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3121 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3181 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3241 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3301 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3361 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3421 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3481 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3541 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3601 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3661 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3721 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3781 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3841 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagatccacg
     3901 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3961 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4021 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4081 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4141 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4201 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4261 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4321 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4381 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4441 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4501 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4561 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4621 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4681 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4741 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     4801 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     4861 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     4921 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     4981 t
//
