LOCUS       sequence-472788-        7709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac integration vector for ComMAND closed-loop circuit 
            regulating mRuby2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7709)
  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 7709)
  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(miRE.FF4)-TS.FF4x1-bGH]-UbC-PuroR-T2A-mGL-SV40pA
FEATURES             Location/Qualifiers
     source          1..7709
                     /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"
     ncRNA           complement(2851..2963)
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(3020..3034)
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     regulatory      3269..3278
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3333..4514)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3342..4284
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3365..3385)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(4688..4707)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        4817..6028
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5865..5884
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             6091..6687
                     /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(6091..6110)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6587..6607
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6697..6750
                     /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(6751..6753,6754..6756,6757..7470)
                     /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(6796..6817)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    7482..7603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7519..7538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7573..7592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7644..7706
                     /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 gcaactcctc
     2101 ctaggtttaa ttaaagactt caagcggcca caagcttgtt gttacttgta cagctcgtcc
     2161 atcccaccac caagcccggc gaacttggca actgcgtgtt cgcgttgtac tacgaacatt
     2221 tcattgtcac tttcctctaa cctttccagg cggtgatcaa cggcatggat accgggcatc
     2281 ttgatgttcc cgacggtctt ttttgacctg taagttgtta cgaaagagca agacagatgg
     2341 ccaccaccat caactttcag tgccatatga gtgtatcccc tcagaccacc atctgctgga
     2401 tacatcatct ctgtattagg ctcccaaccc ttggtcttct tctgcatcac gggaccattg
     2461 gagggaaagt ttacccctct gacttggacg tggtaaacga gacagccatc ctcaaggctg
     2521 gtgtcctgca tgacggtgac gactccacca tcttcgtatc tcgtaactct ttcccaagta
     2581 aaaccctcag gaaaggactg tttaaagaaa tcaggaatgc ctttcgggta cttgataaaa
     2641 gtacggctgc catacatgaa cgacgtggca agaatgtcaa aggcaaatgg caggggtcct
     2701 ccctcgatga ctttgatcct catggtttga gttcccatgt acggattgcc ttctccttca
     2761 cctgtgcatt tgaattggtg gccgttgacc gaaccttcca tgaccacctg tgaaaaaaaa
     2821 aaagaagact cgaagttagt acaattgaaa aaagtgattt aatttatacc attttaattc
     2881 agctttgtaa aaatgtatca aagagatagc aaggtattca gttttagtaa acaagataat
     2941 tgctcctaaa gtagcccctt gaattctagc cccttgaagt ccgaggcagt aggcaccgct
     3001 tgaagtcttt aattaaatac atctgtggct tcactattta attaaagact tcaagcggcg
     3061 ctcactgtca acagcaatat accttctcga gccttctgtt gggttaagaa gtccctgaag
     3121 aagtaatccc agcaagtgtt tccaagatgt gcaggcaacg attctgtaaa gtactgaagc
     3181 ctcattcaac atagtatatg tgctgccgaa gcgagcacac ttaccttcat acgcatattt
     3241 tccttgatca gctcttcgcc cttagacacc atggtggcca ttacagcgtg gatggcgtct
     3301 ccaggcgatc tgacggttca ctaaacgagc tctcacgaca cctgaaatgg aagaaaaaaa
     3361 ctttgaacca ctgtctgagg cttgagaatg aaccaagatc caaactcaaa aagggcaaat
     3421 tccaaggaga attacatcaa gtgccaagct ggcctaactt cagtctccac ccactcagtg
     3481 tggggaaact ccatcgcata aaacccctcc ccccaaccta aagacgacgt actccaaaag
     3541 ctcgagaact aatcgaggtg cctggacggc gcccggtact ccgtggagtc acatgaagcg
     3601 acggctgagg acggaaaggc ccttttcctt tgtgtgggtg actcacccgc ccgctctccc
     3661 gagcgccgcg tcctccattt tgagctccct gcagcagggc cgggaagcgg ccatctttcc
     3721 gctcacgcaa ctggtgccga ccgggccagc cttgccgccc agggcggggc gatacacggc
     3781 ggcgcgaggc caggcaccag agcaggccgg ccagcttgag actacccccg tccgattctc
     3841 ggtggccgcg ctcgcaggcc ccgcctcgcc gaacatgtgc gctgggacgc acgggccccg
     3901 tcgccgcccg cggccccaaa aaccgaaata ccagtgtgca gatcttggcc cgcatttaca
     3961 agactatctt gccagaaaaa aagcgtcgca gcaggtcatc aaaaatttta aatggctaga
     4021 gacttatcga aagcagcgag acaggcgcga aggtgccacc agattcgcac gcggcggccc
     4081 cagcgcccaa gccaggcctc aactcaagca cgaggcgaag gggctcctta agcgcaaggc
     4141 ctcgaactct cccacccact tccaacccga agctcgggat caagaatcac gtactgcagc
     4201 caggggcgtg gaagtaattc aaggcacgca agggccataa cccgtaaaga ggccaggccc
     4261 gcgggaacca cacacggcac ttacctgtgt tctggcggca aacccgttgc gaaaaagaac
     4321 gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag
     4381 ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg caccggttca
     4441 attgccgacc cctcccccca acttctcggg gactgtgggc gatgtgcgct ctgcccactg
     4501 acgggcaccg gagcagtacg aacatggtag ctagtggatc cttcgtcgac ctgcagactg
     4561 gctgtgtata agggagcctg acatttatat tccccagaac atcaggttaa tggcgttttt
     4621 gatgtcattt tcgcggtggc tgagatcagc cacttcttcc ccgataacgg agaccggcac
     4681 actggcgcat atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt
     4741 aaagttcacg catatgaagc tggacatcac ctcccacaac ctcgaggtgg aacagtacgc
     4801 tgatatctct tatactggcc tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc
     4861 ctcacggcga gcgctgccac gtcagacgaa gggcgcagcg agcgtcctga tccttccgcc
     4921 cggacgctca ggacagcggc ccgctgctca taagactcgg ccttagaacc ccagtatcag
     4981 cagaaggaca ttttaggacg ggacttgggt gactctaggg cactggtttt ctttccagag
     5041 agcggaacag gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg
     5101 ggcggtgaac gccgatgatt atataaggac gcgccgggtg tggcacagct agttccgtcg
     5161 cagccgggat ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca cttggtgagt
     5221 agcgggctgc tgggctggcc ggggctttcg tggccgccgg gccgctcggt gggacggaag
     5281 cgtgtggaga gatcgccaag ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg
     5341 ggggttgggg ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt
     5401 gtgaggcggg ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc
     5461 aaggtcttga ggccttcgct aatgcgggaa agctcttatt cgggtgagat gggctggggc
     5521 accatctggg gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg
     5581 ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc
     5641 gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca
     5701 cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg
     5761 gtaggctctc ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg
     5821 tcagtttctt tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg
     5881 aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat
     5941 gtaatcattt gggtcaatat gtaattttca gtgttagact agtaaattgt ccgctaaatt
     6001 ctggccgttt ttggcttttt tgttagacga gctcgtttag tgaaccgtca gatcgcctgg
     6061 agacgccatc cacgctgtaa tgaagccacc atgaccgagt acaagcccac ggtgcgcctc
     6121 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     6181 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     6241 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     6301 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     6361 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     6421 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     6481 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     6541 gccgagcgcg ccggggtgcc cgccttcctg gagacatccg cgccccgcaa cctccccttc
     6601 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     6661 tggtgcatga cccgcaagcc cggtgccgga agcggagaag gtagaggttc tctcctcact
     6721 tgtggtgatg ttgaagaaaa ccctggtcca atggtgagca agggcgagga gctgttcacc
     6781 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtc
     6841 cgcggcgagg gcgagggcga tgccaccaac ggcaagctga ccctgaagtt catctgcacc
     6901 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttaggcta cggcgtggcc
     6961 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     7021 gaaggctacg tccaggagcg caccatctct ttcaaggacg acggtaccta caagacccgc
     7081 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgtgctgaa gggcatcgac
     7141 ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa cagccacaag
     7201 gtctatatca cggccgacaa gcagaagaac ggcatcaagg ctaacttcaa gacccgccac
     7261 aacgttgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     7321 gacggccccg tgctgctgcc cgacaaccac tacctgagcc atcagtccaa actgagcaaa
     7381 gaccccaacg agaagcgcga tcacatggtc ctgaaggaga gggtgaccgc cgccgggatt
     7441 acacatgaca tggacgagct gtacaagtaa caacgggatc caacttgttt attgcagctt
     7501 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     7561 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttacgctcgc tgatataggt
     7621 aggctcgagc agagaagctt acacatgcgt caattttacg catgattatc tttaacgtac
     7681 gtcacaatat gattatcttt ctagggtta
//
