LOCUS       sequence-477089-        4301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI Golden Gate assembly plasmid encoding the COMET 
            NLS-FXBP-ZF43-P2A-NES-VP64-FRB-P2A-iRFP720 CDS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4301)
  AUTHORS   Peterman EL, Ploessl DS, Love KS, Sanabria V, Daniels RF, Johnstone 
            CP, Godavarti DR, Kabaria SR, Oakes CG, Pai AA, Galloway KE
  TITLE     High-resolution profiling reveals coupled transcriptional and 
            translational regulation of transgenes.
  JOURNAL   Nucleic Acids Research, Volume 53, Issue 11, 24 June 2025, gkaf528
  PUBMED    39651241
REFERENCE   2  (bases 1 to 4301)
  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: "Nucleic 
            Acids Research, Volume 53, Issue 11, 24 June 2025, gkaf528"
COMMENT     Sequence Label: 
            pKG3658_pPV2-NLS-FXBP-ZF43-P2A-NES-VP64-FRB-P2A-iRFP720
FEATURES             Location/Qualifiers
     source          1..4301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      41..629
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     530..549
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      654..663
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             663..728
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             735..755
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(798..800,801..1121)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1458..1514
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1602..1751
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             2154..2210
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2211..3161
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 720 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP720
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     promoter        3206..3310
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3311..3379,3380..4171)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGYRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3529..3548)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
       61 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
      121 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
      181 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
      241 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
      301 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
      361 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
      421 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      481 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      541 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
      601 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgggtctcaa atggccacca
      661 tggattataa ggatcacgat ggcgactaca aggaccacga tattgactac aaagacgatg
      721 acgataaaat ggctcctaag aaaaagcgca aagtcggtat ccatggcgtt cccgtcgacg
      781 gtggactcca agttaagatg ggagtgcagg tggaaaccat ctccccagga gacgggcgca
      841 ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt gaagatggaa
      901 agaaatttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta ggcaagcagg
      961 aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag agagccaaac
     1021 tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc atcccaccac
     1081 atgccactct cgtctttgat gtggagcttc taaaactgga aggatccggt ggtggcgggt
     1141 ctggaggagg aggtagtggt ggaggtggct ctggtaccgc tagacccggg gagcgcccct
     1201 tccagtgtcg catttgcatg cggaactttt cgcgccagga caggcttgac aggcataccc
     1261 gtactcatac cggtgaaaaa ccgtttcagt gtcggatctg tatgcgaaat ttctcccaga
     1321 aggagcactt ggcggggcat ctacgtacgc acaccggcga gaagccattc caatgccgaa
     1381 tatgcatgcg caacttcagt cgccgcgaca acctgaaccg gcacctaaaa acccacctga
     1441 gaggatctgg aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg
     1501 agaaccctgg acctgcacca ggaagtatgg aggaactttc acaggccctg gctagtagtt
     1561 tttcagtgtc tggtgtcggt atccatggcg ttcccggatc agacgcattg gacgattttg
     1621 atctggatat gctgggaagt gacgccctcg atgattttga ccttgacatg cttggttcgg
     1681 atgcccttga tgactttgac ctcgacatgc tcggcagtga cgcccttgat gatttcgacc
     1741 tggacatgct gattaactct agaagttccg gatcttctgg ttctggttct ggttcaggat
     1801 ccggtggtgg cgggtctgga ggaggaggta gtggtggagg tggctctggt accatgcttg
     1861 agatgtggca tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa aggaacgtga
     1921 aaggcatgtt tgaggtgctg gagcccttgc atgctatgat ggaacgggga ccccagactc
     1981 tgaaggaaac atcctttaat caggcctatg gtcgagattt aatggaggcc caagagtggt
     2041 gcaggaagta catgaaatca gggaatgtca aggacctcct ccaagcctgg gacctctatt
     2101 atcatgtgtt ccgacgaatc tcaaagacta gagctagcgg ccgcggaagc ggagctacta
     2161 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct atggcggaag
     2221 gatccgtcgc caggcagcct gacctcttga cctgcgacga tgagccgatc catatccccg
     2281 gtgccatcca accgcatgga ctgctgctcg ccctcgccgc cgacatgacg atcgttgccg
     2341 gcagcgacaa ccttcccgaa ctcaccggac tggcgatcgg cgccctgatc ggccgctctg
     2401 cggccgatgt cttcgactcg gagacgcaca accgtctgac gatcgccttg gccgagcccg
     2461 gggcggccgt cggagcaccg atcactgtcg gcttcacgat gcgaaaggac gcaggcttca
     2521 tcggctcctg gcatcgccat gatcagctca tcttcctcga gctcgagcct ccccagcggg
     2581 acgtcgccga gccgcaggcg ttcttccgcc gcaccaacag cgccatccgc cgcctgcagg
     2641 ccgccgaaac cttggaaagc gcctgcgccg ccgcggcgca agaggtgcgg aagattaccg
     2701 gcttcgatcg ggtgatgatc tatcgcttcg cctccgactt cagcgggtcc gtgatcgcag
     2761 aggatcggtg cgccgaggtc gagtcaaaac taggcctgca ctatcctgcc tcattcatcc
     2821 cggcgcaggc ccgtcggctc tataccatca acccggtacg gatcattccc gatatcaatt
     2881 atcggccggt gccggtcacc ccagacctca atccggtcac cgggcggccg attgatctta
     2941 gcttcgccat cctgcgcagc gtctcgccca accatctgga gttcatgcgc aacataggca
     3001 tgcacggcac gatgtcgatc tcgattttgc gcggcgagcg actgtgggga ttgatcgttt
     3061 gccatcaccg aacgccgtac tacgtcgatc tcgatggccg ccaagcctgc gagctagtcg
     3121 cccaggttct ggcctggcag atcggcgtga tggaagagta acaacagaga cctagacgtc
     3181 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3241 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3301 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3361 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3421 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3481 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3541 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3601 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3661 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3721 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3781 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3841 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3901 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3961 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4021 gcgtgggtat cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4081 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4141 gataggtgcc tcactgatta agcattggta actgtcatac caagtttact catatatact
     4201 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4261 taatctcatg accaaaatcc cttaacgtga gttttcgttc c
//
