LOCUS       sequence-318789-        5446 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression ofCMV 3xFLAG-NLS-DsDed-ZF1 under the CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5446)
  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 5446)
  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: pJB002 CMV 3xFLAG-NLS-DsDed-ZF1
FEATURES             Location/Qualifiers
     source          1..5446
                     /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"
     CDS             811..1485
                     /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(991..1011)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1403..1426
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1842..1859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1848..2072
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2118..2546
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2205..2224)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2415..2436
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2569..2765
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2616..2751
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2678..2697
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2885..3006
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2922..2941)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2976..2995
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3055..3071)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3055..3071)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3068..3090)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3079..3095
                     /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(3103..3109,3110..3127,3128..3133))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3148..3169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3286..3303)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3457..4045)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3537..3556)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4216..5007,5008..5076))
                     /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     4839..4858
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5077..5181)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5256..5275)
                     /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 caggctccgg aagtggctcc atggatagca ctgagaacgt catcaagccc
      841 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
      901 ggcgagggcg agggcaagcc ctacgagggc acccagaccg ccaagctcca ggtgaccaag
      961 ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttccagta cggctccaag
     1021 gtgtacgtga agcaccccgc cgacatcccc gactacaaga agctgtcctt ccccgagggc
     1081 ttcaagtggg agaaggtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     1141 tcctccctcc aggacggcac cttcatctac cacgtgaagt tcatcggcgt gaacttcccc
     1201 tccgacggcc ccgtaatgca gaagaaaact ctgggctggg agccctccac cgagcgcctg
     1261 tacccccgcg acggcgtgct gaagggcgag atccacaagg cgctgaagct gaagggcggc
     1321 ggccactacc tggtggagtt caagtcaatc tacatggcca agaagcccgt gaagctgccc
     1381 ggctactact acgtggactc caagctggac atcacctccc acaacgagga ctacaccgtg
     1441 gtggagcagt acgagcgcgc cgaggcccgc caccacctgt tccagtctgg ttctggttct
     1501 ggttcaggat ccggtaccgg tggagctaga cccggggagc gccccttcca gtgtcgcatt
     1561 tgcatgcgga acttttcgcg ccaggacagg cttgacaggc atacccgtac tcataccggt
     1621 gaaaaaccgt ttcagtgtcg gatctgtatg cgaaatttct cccagaagga gcacttggcg
     1681 gggcatctac gtacgcacac cggcgagaag ccattccaat gccgaatatg catgcgcaac
     1741 ttcagtcgcc gcgacaacct gaaccggcac ctaaaaaccc acctgagagg atcttaagcg
     1801 gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     1861 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     1921 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     1981 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagtcaatag
     2041 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2101 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2161 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2221 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2281 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2341 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2401 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2461 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2521 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt aggctcaatt
     2581 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     2641 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     2701 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     2761 gcaaaaagct cccgggagct tgtatatcca ttttcggagg ttgggcttcg gaatcgtttt
     2821 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     2881 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2941 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3001 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     3061 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3121 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3181 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3241 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3301 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3361 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3421 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3481 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3541 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3601 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3661 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3721 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3781 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3841 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3901 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3961 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4021 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4081 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4141 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4201 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4261 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4321 cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga
     4381 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4441 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4501 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4561 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4621 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4681 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4741 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4801 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4861 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4921 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4981 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5041 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5101 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5161 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     5221 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5281 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     5341 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     5401 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgtt
//
