LOCUS       sequence-274359-        8064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac vector for constitutive NanoLuc expression in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8064)
  AUTHORS   Chu LF, Mamott D, Ni Z, Bacher R, Liu C, Swanson S, Kendziorski C, 
            Stewart R, Thomson JA
  TITLE     An In Vitro Human Segmentation Clock Model Derived from Embryonic 
            Stem Cells.
  JOURNAL   Cell Rep. 2019 Aug 27;28(9):2247-2255.e5. doi: 
            10.1016/j.celrep.2019.07.090.
  PUBMED    31461642
REFERENCE   2  (bases 1 to 8064)
  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"; doi: 
            "10.1016/j.celrep.2019.07"; journalName: "Cell Rep"; date: 
            "2019-08-27- 27"; volume: "28"; issue: "9"; pages: "2247-2255.e5"
COMMENT     Sequence Label: pB-EF1a-NLuc-IRES-Puro
FEATURES             Location/Qualifiers
     source          1..8064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   418..480
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1423..2585
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1654..2576
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2533..2553
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2635..3150
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     intron          3205..3434
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3417..3436
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3492..4065
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3659..3676)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3886..3905
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4098..4697
                     /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(4098..4117)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4594..4614
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4827..4961
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4851..4870)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4905..4924
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(5432..5465)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     repeat_region   5783..5817
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5843..5859)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5843..5859)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5856..5878)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5867..5883
                     /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(5891..5897,5898..5915,5916..5921))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5936..5957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6074..6091)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6245..6833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6325..6344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7004..7795,7796..7864))
                     /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     7627..7646
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7865..7969)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8037..8055
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt caagctttat tatttaaccc
      421 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
      481 tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat
      541 tatatttaca cttacatact aataataaat tcaacaaaca atttatttat gtttatttat
      601 ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttaaacat
      661 tctctctttt acaaaaataa acttattttg tactttaaaa acagtcatgt tgtaatcgat
      721 aataagtaat tagcttaacc tatacataat agaaacaaat tatacttatt agtcagtcag
      781 aaacaacttt ggcacatatc aatattatgc tctcgttaat taactcgagg gatccggcgc
      841 gcctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt
      901 ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc
      961 cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg
     1021 agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc
     1081 tgggggcttt gggagggggc tgtccccgtg agctcccagg cgcgcctgtc attctaaatc
     1141 tctctttcag cctaaagctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
     1201 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
     1261 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
     1321 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
     1381 ggctgtcccc gtgagctccc aggcgcgccg ctagcttcgt gaggctccgg tgcccgtcag
     1441 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     1501 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     1561 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     1621 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
     1681 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctccag
     1741 tacgtgattc ttgatcccga gctggagcca ggggcgggcc ttgcgcttta ggagcccctt
     1801 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
     1861 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
     1921 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caggatctgc
     1981 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
     2041 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
     2101 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
     2161 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
     2221 ctgctccagg gggctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac
     2281 ccacacaaag gaaaggggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     2341 accgggcgcc gtccaggcac ctcgattagt tctggagctt ttggagtacg tcgtctttag
     2401 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
     2461 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat
     2521 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt
     2581 cgtgaacacg tggtcgcggc caattctgca gtcgagctca agcttcgaac caccatggtc
     2641 ttcacactcg aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa
     2701 gtccttgaac agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg
     2761 atccaaagga ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc
     2821 ccgtatgaag gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg
     2881 taccctgtgg atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac
     2941 ggggttacgc cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc
     3001 gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag
     3061 cgcctgatca accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc
     3121 tggcggctgt gcgaacgcat tctggcgtaa gcggccgcga ctctactgga attaattcgc
     3181 tgtctgcgag ggccagctgt tggggtgagt actccctctc aaaagcgggc atgacttctg
     3241 cgctaagatt gtcagtttcc aaaaacgagg aggatttgat attcacctgg cccgcggtga
     3301 tgcctttgag ggtggccgcg tccatctggt cagaaaagac aatctttttg ttgtcaagct
     3361 tgaggtgtgg caggcttgag atctggccat acacttgagt gacaatgaca tccactttgc
     3421 ctttctctcc acaggtgtcc actcccaggt ccaactgcag gtcgagcatg catctagggc
     3481 ggccaattcc gcccctctcc cccccccccc cctaacgtta ctggccgaag ccgcttggaa
     3541 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
     3601 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
     3661 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     3721 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     3781 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     3841 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     3901 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     3961 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
     4021 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagcttg ccacaaccca
     4081 caaggagacg accttccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     4141 acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     4201 acaccgtcga cccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     4261 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     4321 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     4381 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     4441 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     4501 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     4561 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     4621 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     4681 gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     4741 gaccccatgg ctccgaccga agccacccgg ggcggccccg ccgaccccgc acccgccccc
     4801 gaggcccacc gactctagag ctcgctgatc cagacatgat aagatacatt gatgagtttg
     4861 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     4921 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     4981 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
     5041 acaaatgtgg tatggctgat tatgatccgg ctgcctcgcg cttaattaac ccgggggccg
     5101 cctgggaact gccgctggcc ccccaccgcc ccaaggatct ccggtccccg cccggcttgc
     5161 tgacgtcacg gatgccccag ggtgtgctgg gcaggtagcg ggagcctggg aaatggagtc
     5221 cattagcaga agtggccact tccaggagtc gctgtgcccc gatgcacact gggaagtccg
     5281 cagctccgag gcccccagtg gaaatcgcca gatgagggcc tcctccgggg aatgctggga
     5341 aatggagtct acaggccgga ggggtcccca cggcatacta gcaagtgtgt gtagcaccgc
     5401 gtaaagggga tgaatagcag actgccccgg gataacttcg tataggatac tttatacgaa
     5461 gttatgagct ctgtgataat cactagtgaa ttgatactag tcggtcacga tatctataac
     5521 aagaaaatat atatataata agttatcacg taagtagaac atgaaataac aatataatta
     5581 tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg
     5641 cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag
     5701 ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag
     5761 agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta
     5821 aaaaaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5881 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5941 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6001 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6061 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6121 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6181 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6241 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6301 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6361 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6421 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6481 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6541 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6601 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6661 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6721 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6781 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6841 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6901 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6961 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7021 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7081 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7141 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7201 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7261 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7321 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7381 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7441 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7501 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7561 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7621 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7681 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7741 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7801 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7861 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7921 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7981 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     8041 ggcgtatcac gaggcccttt cgtc
//
