LOCUS       sequence-223037-        4774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac transposon plasmid CRISPR gene deletion activatable 
            fluorescence. Constitutive iRFP670 under EF1A promoter, CMV promoter
            with two SV40 polyA followed by red-enhanced nanolantern (ReNL).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4774)
  AUTHORS   Rui Y, Wilson DR, Choi J, Varanasi M, Sanders K, Karlsson J, Lim M, 
            Green JJ
  TITLE     Carboxylated branched poly(beta-amino ester) nanoparticles enable 
            robust cytosolic protein delivery and CRISPR-Cas9 gene editing.
  JOURNAL   Sci Adv. 2019 Dec 6;5(12):eaay3255. doi: 10.1126/sciadv.aay3255. 
            eCollection 2019 Dec.
  PUBMED    31840076
REFERENCE   2  (bases 1 to 4774)
  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: "2019-12-6"; volume: "5(12):eaay3255. doi"; pages: " 
            10.1126/sciadv.aay3255. eCollection 2019 Dec"
COMMENT     Sequence Label: PB-iRFP-STOP-ReNL
FEATURES             Location/Qualifiers
     source          1..4774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    253..276
                     /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(280..286,287..304,305..310))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    325..346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        405..600
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      451..586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    606..740
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(643..662)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     697..716
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(808..825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(979..1567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1059..1078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1738..2529,2530..2598))
                     /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     2361..2380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2599..2670)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2821..2832)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        3063..3101
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3098..3122
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     LTR             3228..3496
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3548..3557
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3554..3556,3557..3559,3560..4273)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3554..3556,3557..3559,3560..4270)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3599..3620)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3860..3879)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4207..4228
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4316..4537
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(4645..4656)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 gttatatagg tcttagccaa aacatgtgat agtcactcca ggggttgctg gaaaagaagt
       61 ctgtgacact cattaaccta ttggtgcaga atttgaatga tctaaaggag acgcatgcaa
      121 gcttttctta agtgatcaag cagcccgtgc aagcttttct taagtgatca agcagcccgt
      181 gcaagctttt cttaagtgat caagcagccc gtgcaagctt ttcttaagtg atcaagcagc
      241 ccgtgcctgt gtgaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
      301 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
      361 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caataagtca
      421 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
      481 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
      541 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
      601 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
      661 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
      721 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      781 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
      841 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
      901 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      961 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     1021 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     1081 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     1141 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1201 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1261 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1321 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1381 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     1441 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1501 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1561 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     1621 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1681 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1741 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     1801 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1861 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     1921 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1981 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     2041 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     2101 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     2161 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2221 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2281 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2341 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2401 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2461 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     2521 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aatggaataa gggcgacacg
     2581 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     2641 ttgtctcatg agcggataca tatttgaatg tatttagaaa aatccgtgag cagcaggatc
     2701 ctgtacatac atgtgcatcg acctggtgac ctttattcat aagagatgat gtattcttga
     2761 tactacttct catttgcaaa ttccaattat tattaatttc atatcaatta gaataatata
     2821 tcttccttca atttagttac ctcactatgg gctatgtaca aactccaaga aaaagttagt
     2881 catgtgcttt gcagaagata aaagcttagt gtaaaacagg ctgagagtat ttgatgtaag
     2941 aaggggagtg gttatatagg tcttagccaa aacatgtgat agtcactcca ggggttgctg
     3001 gaaaagaagt ctgtgacact cattaaccta ttggtgcaga atttgaatga tctaaaggag
     3061 acggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg aaccgtcaga
     3121 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
     3181 gcctccgcgg ccccgcattc gagctcggta cccggcaatt gcctgcgggg gctcgcatct
     3241 ctccttcacg cgcccgccgc cctacctgag gccgccatcc acgccggttg agtcgcgttc
     3301 tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc
     3361 tcaggtcgag accgggcctt tgtccggcgc tcccttggag cctacctaga ctcagccggc
     3421 tctccacgct ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg ttttctgttc
     3481 tgcgccgtta cagatcatct cgagaattct gcagtcgacg gtaccgcggg cccgggatcc
     3541 accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     3601 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     3661 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3721 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     3781 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3841 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3901 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3961 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     4021 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     4081 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     4141 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     4201 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     4261 gctgtacaag taaagcggcc gcttcacatg caccggtaat gatcactagt gtcgactgtg
     4321 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     4381 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     4441 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     4501 gacaatagca ggcatgggga tgcggtgggc tctatgggcc cgggcgatat cgacctttat
     4561 tcataagaga tgatgtattc ttgatactac ttctcatttg caaattccaa ttattattaa
     4621 tttcatatca attagaataa tatatcttcc ttcaatttag ttacctcact atgggctatg
     4681 tacaaactcc aagaaaaagt tagtcatgtg ctttgcagaa gataaaagct tagtgtaaaa
     4741 caggctgaga gtatttgatg taagaagggg agtg
//
