LOCUS       sequence-318364-        4900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-intC-ZF2 under the CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4900)
  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 4900)
  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: pJB005 CMV 3xFLAG-NLS-intC-ZF2
FEATURES             Location/Qualifiers
     source          1..4900
                     /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(1296..1313)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1302..1526
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1572..2000
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1659..1678)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1869..1890
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2023..2219
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2070..2205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2132..2151
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2339..2460
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2376..2395)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2430..2449
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2509..2525)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2509..2525)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2522..2544)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2533..2549
                     /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(2557..2563,2564..2581,2582..2587))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2602..2623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2740..2757)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2911..3499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2991..3010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3670..4461,4462..4530))
                     /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     4293..4312
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4531..4635)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4710..4729)
                     /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 aatgatgctg aagaagatcc tgaagatcga ggagctggac
      841 gagagagagc tgatcgacat cgaggtgagc ggcaaccacc tgttctacgc caacgacatc
      901 ctgacccaca acagcaacag gggtggtggc gggtctggtg gaggaggtag tggtggaggt
      961 ggctctggta ccggtggagc tagacccggg gagcgcccct tccagtgtcg catttgcatg
     1021 cggaactttt cgagaaattt cattttgcaa agacataccc gtactcatac cggtgaaaaa
     1081 ccgtttcagt gtcggatctg tatgcgaaat ttctccgata gagctaattt gaggaggcat
     1141 ctgcgtacgc acaccggcga gaagccattc caatgccgaa tatgcatgcg caacttcagt
     1201 agacatgatc aattgactag acacctaaaa acccacctga gaggatctta agcggccgct
     1261 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     1321 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1381 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     1441 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagtca atagcaggca
     1501 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     1561 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1621 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1681 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1741 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1801 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1861 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1921 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1981 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttaggctc aattagtcag
     2041 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     2101 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     2161 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     2221 agctcccggg agcttgtata tccattttcg gaggttgggc ttcggaatcg ttttccggga
     2281 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     2341 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2401 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2461 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     2521 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2581 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2641 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2701 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2761 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2821 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2881 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2941 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3001 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3061 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3121 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3181 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3241 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3301 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3361 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3421 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3481 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3541 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3601 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3661 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3721 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3781 atctggcccc agtgctgcaa tgataccgcg agatccacgc tcaccggctc cagatttatc
     3841 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3901 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3961 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4021 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4081 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4141 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4201 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4261 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4321 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4381 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4441 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4501 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4561 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4621 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     4681 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4741 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     4801 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     4861 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
//
