LOCUS       sequence-474133-        7250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac integration vector for ComMAND base gene regulating mRuby2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7250)
  AUTHORS   Love KS, Johnstone CP, Peterman EL, Gaglione S, Birnbaum ME, 
            Galloway KE
  TITLE     Model-guided design of microRNA-based gene circuits supports precise
            dosage of transgenic cargoes into diverse primary cells.
  JOURNAL   Cell Syst. 2025 Apr 22:101269. doi: 10.1016/j.cels.2025.101269.
  PUBMED    40300600
REFERENCE   2  (bases 1 to 7250)
  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: "Cell Syst. 
            2025 Apr 22:101269. doi: 10.1016/j.cels.2025.101269."
COMMENT     Sequence Label: pPB-[EF1a-mRuby2-bGH]-UbC-PuroR-T2A-mGL-SV40pA
FEATURES             Location/Qualifiers
     source          1..7250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(116..184,185..976)
                     /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     complement(334..353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1147..1735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1636..1655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1817..1851
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1864..2088
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(2103..2813)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     regulatory      2810..2819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2874..4055)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2883..3825
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2906..2926)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(4229..4248)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        4358..5569
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5406..5425
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5632..6228
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5632..5651)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6128..6148
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6238..6291
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6292..6294,6295..6297,6298..7011)
                     /codon_start=1
                     /product="monomeric GFP derivative that displays 
                     exceptionally high brightness in cells (Campbell et al., 
                     2020)"
                     /label=mGreenLantern
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIVLKGIDFKEDGNILGHKLEYNFNSHKVYITADKQKNGIK
                     ANFKTRHNVEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLK
                     ERVTAAGITHDMDELYK"
     primer_bind     complement(6337..6358)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    7023..7144
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7060..7079)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7114..7133
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7185..7247
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 ctataatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
       61 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
      121 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
      181 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
      241 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
      301 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
      361 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
      421 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
      481 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
      541 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
      601 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
      661 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
      721 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
      781 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
      841 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
      901 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
      961 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     1021 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     1081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1141 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1201 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1261 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     1321 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     1381 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     1441 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     1501 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     1561 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     1621 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     1681 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     1741 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     1801 tcctgcgtta tcttcaccct agaaagatag tctgcgtaaa attgacgcat gtccttacta
     1861 gcgccataga gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct
     1921 tgctgtcctg ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg
     1981 caatttcctc attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag
     2041 gcacggggga ggggcaaaca acagatggct ggcaactaga aggcacaggt gcaacgttgt
     2101 tacttgtaca gctcgtccat cccaccacca agcccggcga acttggcaac tgcgtgttcg
     2161 cgttgtacta cgaacatttc attgtcactt tcctctaacc tttccaggcg gtgatcaacg
     2221 gcatggatac cgggcatctt gatgttcccg acggtctttt ttgacctgta agttgttacg
     2281 aaagagcaag acagatggcc accaccatca actttcagtg ccatatgagt gtatcccctc
     2341 agaccaccat ctgctggata catcatctct gtattaggct cccaaccctt ggtcttcttc
     2401 tgcatcacgg gaccattgga gggaaagttt acccctctga cttggacgtg gtaaacgaga
     2461 cagccatcct caaggctggt gtcctgcatg acggtgacga ctccaccatc ttcgtatctc
     2521 gtaactcttt cccaagtaaa accctcagga aaggactgtt taaagaaatc aggaatgcct
     2581 ttcgggtact tgataaaagt acggctgcca tacatgaacg acgtggcaag aatgtcaaag
     2641 gcaaatggca ggggtcctcc ctcgatgact ttgatcctca tggtttgagt tcccatgtac
     2701 ggattgcctt ctccttcacc tgtgcatttg aattggtggc cgttgaccga accttccatg
     2761 accaccttca tacgcatatt ttccttgatc agctcttcgc ccttagacac catggtggcc
     2821 attacagcgt ggatggcgtc tccaggcgat ctgacggttc actaaacgag ctctcacgac
     2881 acctgaaatg gaagaaaaaa actttgaacc actgtctgag gcttgagaat gaaccaagat
     2941 ccaaactcaa aaagggcaaa ttccaaggag aattacatca agtgccaagc tggcctaact
     3001 tcagtctcca cccactcagt gtggggaaac tccatcgcat aaaacccctc cccccaacct
     3061 aaagacgacg tactccaaaa gctcgagaac taatcgaggt gcctggacgg cgcccggtac
     3121 tccgtggagt cacatgaagc gacggctgag gacggaaagg cccttttcct ttgtgtgggt
     3181 gactcacccg cccgctctcc cgagcgccgc gtcctccatt ttgagctccc tgcagcaggg
     3241 ccgggaagcg gccatctttc cgctcacgca actggtgccg accgggccag ccttgccgcc
     3301 cagggcgggg cgatacacgg cggcgcgagg ccaggcacca gagcaggccg gccagcttga
     3361 gactaccccc gtccgattct cggtggccgc gctcgcaggc cccgcctcgc cgaacatgtg
     3421 cgctgggacg cacgggcccc gtcgccgccc gcggccccaa aaaccgaaat accagtgtgc
     3481 agatcttggc ccgcatttac aagactatct tgccagaaaa aaagcgtcgc agcaggtcat
     3541 caaaaatttt aaatggctag agacttatcg aaagcagcga gacaggcgcg aaggtgccac
     3601 cagattcgca cgcggcggcc ccagcgccca agccaggcct caactcaagc acgaggcgaa
     3661 ggggctcctt aagcgcaagg cctcgaactc tcccacccac ttccaacccg aagctcggga
     3721 tcaagaatca cgtactgcag ccaggggcgt ggaagtaatt caaggcacgc aagggccata
     3781 acccgtaaag aggccaggcc cgcgggaacc acacacggca cttacctgtg ttctggcggc
     3841 aaacccgttg cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc
     3901 accctcggga aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca
     3961 ccttctctag gcaccggttc aattgccgac ccctcccccc aacttctcgg ggactgtggg
     4021 cgatgtgcgc tctgcccact gacgggcacc ggagcagtac gaacatggta gctagtggat
     4081 ccttcgtcga cctgcagact ggctgtgtat aagggagcct gacatttata ttccccagaa
     4141 catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc
     4201 cccgataacg gagaccggca cactggcgca tatcggtggt catcatgcgc cagctttcat
     4261 ccccgatatg caccaccggg taaagttcac gcatatgaag ctggacatca cctcccacaa
     4321 cctcgaggtg gaacagtacg ctgatatctc ttatactggc ctccgcgccg ggttttggcg
     4381 cctcccgcgg gcgcccccct cctcacggcg agcgctgcca cgtcagacga agggcgcagc
     4441 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg
     4501 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg
     4561 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat
     4621 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt
     4681 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc
     4741 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg
     4801 ggccgctcgg tgggacggaa gcgtgtggag agatcgccaa gggctgtagt ctgggtccgc
     4861 gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg
     4921 agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg
     4981 catggtgggc ggcaagaacc caaggtcttg aggccttcgc taatgcggga aagctcttat
     5041 tcgggtgaga tgggctgggg caccatctgg ggaccctgac gtgaagtttg tcactgactg
     5101 gagaactcgg tttgtcgtct gttgcggggg cggcagttat ggcggtgccg ttgggcagtg
     5161 cacccgtacc tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc
     5221 ttataatgca gggtggggcc acctgccggt aggtgtgcgg taggcttttc tccgtcgcag
     5281 gacgcagggt tcgggcctag ggtaggctct cctgaatcga caggcgccgg acctctggtg
     5341 aggggaggga taagtgaggc gtcagtttct ttggtcggtt ttatgtacct atcttcttaa
     5401 gtagctgaag ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt
     5461 tttttaggca ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc agtgttagac
     5521 tagtaaattg tccgctaaat tctggccgtt tttggctttt ttgttagacg agctcgttta
     5581 gtgaaccgtc agatcgcctg gagacgccat ccacgctgta atgaagccac catgaccgag
     5641 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     5701 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     5761 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     5821 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     5881 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     5941 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     6001 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     6061 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacatcc
     6121 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     6181 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg aagcggagaa
     6241 ggtagaggtt ctctcctcac ttgtggtgat gttgaagaaa accctggtcc aatggtgagc
     6301 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     6361 aacggccaca agttcagcgt ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg
     6421 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     6481 accttaggct acggcgtggc ctgcttcgcc cgctaccccg accacatgaa gcagcacgac
     6541 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctc tttcaaggac
     6601 gacggtacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     6661 atcgtgctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     6721 tacaacttca acagccacaa ggtctatatc acggccgaca agcagaagaa cggcatcaag
     6781 gctaacttca agacccgcca caacgttgag gacggcggcg tgcagctcgc cgaccactac
     6841 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     6901 catcagtcca aactgagcaa agaccccaac gagaagcgcg atcacatggt cctgaaggag
     6961 agggtgaccg ccgccgggat tacacatgac atggacgagc tgtacaagta acaacgggat
     7021 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     7081 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     7141 cttacgctcg ctgatatagg taggctcgag cagagaagct tacacatgcg tcaattttac
     7201 gcatgattat ctttaacgta cgtcacaata tgattatctt tctagggtta
//
