LOCUS       sequence-256385-        7728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for constitutive expression of EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7728)
  AUTHORS   Strubberg AM, Veronese Paniagua DA, Zhao T, Dublin L, Pritchard T, 
            Bayguinov PO, Fitzpatrick JAJ, Madison BB
  TITLE     The Zinc Finger Transcription Factor PLAGL2 Enhances Stem Cell Fate 
            and Activates Expression of ASCL2 in Intestinal Epithelial Cells.
  JOURNAL   Stem Cell Reports. 2018 Aug 14;11(2):410-424. doi: 
            10.1016/j.stemcr.2018.06.009. Epub 2018 Jul 12.
  PUBMED    30017821
REFERENCE   2  (bases 1 to 7728)
  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.stemcr.2018.06.009"; journalName: "Stem Cell Reports"; 
            date: "2018-08-14- 14"; volume: "11"; issue: "2"; pages: "410-424"
COMMENT     Sequence Label: BII-ChPtW-EGFP
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..39
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(308..324)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(308..324)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1875..1928
                     /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             1935..1961
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1935..1961
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1968..1970,1971..1973,1974..2687)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2013..2034)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2274..2293)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2621..2642
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2705..3293
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2758..2778)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3176..3187)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3373..3507
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3397..3416)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3451..3470
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4285..4347
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4477..4494)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4477..4493)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4486..4508)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4635..5090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4722..4741)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4932..4953
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5116..5220
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5221..5289,5290..6081)
                     /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(5439..5458)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6252..6840
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6741..6760
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6994..7011
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7128..7149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7164..7169,7170..7187,7188..7194)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7202..7218
                     /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)."
     primer_bind     7207..7229
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7226..7242
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7226..7242
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
       61 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
      121 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
      181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
      241 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
      301 tgttatagat ccactagttc tagagggaca gccccccccc aaagccccca gggatgtaat
      361 tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc
      421 gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg
      481 cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg
      541 ggatacgggg aaaaggcctc caaggcctgc aggagtcaat gggaaaaacc cattggagcc
      601 aagtacactg actcaatagg gactttccat tgggttttgc ccagtacata aggtcaatag
      661 ggggtgagtc aacaggaaag tcccattgga gccaagtaca ttgagtcaat agggactttc
      721 caatgggttt tgcccagtac ataaggtcaa tgggaggtaa gccaatgggt ttttcccatt
      781 actgacatgt atactgagtc attagggact ttccaatggg ttttgcccag tacataaggt
      841 caataggggt gaatcaacag gaaagtccca ttggagccaa gtacactgag tcaataggga
      901 ctttccattg ggttttgccc agtacaaaag gtcaataggg ggtgagtcaa tgggtttttc
      961 ccattattgg cacatacata aggtcaatag gggtgactag tggagaagag catgcttgag
     1021 ggctgagtgc ccctcagtgg gcagagagca catggcccac agtccctgag aagttggggg
     1081 gaggggtggg caattgaact ggtgcctaga gaaggtgggg cttgggtaaa ctgggaaagt
     1141 gatgtggtgt actggctcca cctttttccc cagggtgggg gagaaccata tataagtgca
     1201 gtagtctctg tgaacattcg gtttagtgaa cctgcaggtc ggccgccacg accggtgcca
     1261 ccatgactga atacaaacca actgttcgcc tggcaactcg tgatgatgtt ccacgtgcag
     1321 ttcgcaccct ggctgctgca tttgctgact accctgcaac ccgtcacact gtggacccag
     1381 accgccacat tgaacgtgtg actgaactgc aggagctgtt cctgacccgt gtgggcctgg
     1441 acattggcaa agtgtgggtg gcagatgatg gtgctgctgt ggcagtgtgg accacccctg
     1501 aatctgttga agctggtgca gtgtttgctg agattggccc acgcatggca gaactgtctg
     1561 gcagccgcct ggcagcacaa cagcagatgg aaggtctgct ggcaccacac cgcccaaaag
     1621 aacctgcttg gttcctggca actgtgggtg tgagccctga ccaccagggt aagggcctgg
     1681 gctctgcagt ggtgctgcct ggtgtggaag cagctgaacg tgcaggtgtg cctgctttcc
     1741 tggagacctc agctccacgc aacctgcctt tctatgaacg cctgggcttc actgtgactg
     1801 ctgatgtgga agtgccagaa ggcccacgca cttggtgcat gactcgcaaa ccaggtgcta
     1861 gccttggctc cggcgagggc aggggaagtc ttctaacatg cggggacgtg gaggaaaatc
     1921 ccggcccact cggctacccc tacgacgtgc ccgactacgc cagcggcatg gtgagcaagg
     1981 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2041 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     2101 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct
     2161 tcacctacgg cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct
     2221 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     2281 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2341 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2401 actacaacag ccacaaggtc tatatcaccg ccgacaagca gaagaacggc atcaaggtga
     2461 acttcaagac ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     2521 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     2581 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     2641 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagag ttcgcgtctg
     2701 gaacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     2761 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     2821 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     2881 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     2941 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3001 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     3061 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
     3121 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     3181 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     3241 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggaat
     3301 taattctggc taagcacttc gtggccgtcg atcgtttaaa gggaggtagt gagtcgagat
     3361 atcgatacta gtgatccaga catgataaga tacattgatg agtttggaca aaccacaact
     3421 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3481 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3541 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     3601 gctgattatg atcctctaga gggacagccc ccccccaaag cccccaggga tgtaattacg
     3661 tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc
     3721 cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg
     3781 ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat
     3841 acggggaaaa gccataccaa tggagctcac ggggacagcc cccccccaaa gcccccaggg
     3901 atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg
     3961 gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg
     4021 aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca
     4081 cctgggggga tacggggaaa aagaattcga taaaagtttt gttactttat agaagaaatt
     4141 ttgagttttt gtttttttta ataaataaat aaacataaat aaattgtttg ttgaatttat
     4201 tattagtatg taagtgtaaa tataataaaa cttaatatct attcaaatta ataaataaac
     4261 ctcgatatac agaccgataa aacacatgcg tcaattttac acatgattat ctttaacgta
     4321 cgtcacaata tgattatctt tctagggtta atctagctgc gtgttctgca gcgtgtcgag
     4381 catcttcatc tgctccatca cgctgtaaaa cacatttgca ccgcgagtct gcccgtcctc
     4441 cacgggttca aaaacgtgaa tgaacgaggc gcgctcactg gccgtcgttt tacaacgtcg
     4501 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4561 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4621 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4681 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4741 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4801 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4861 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4921 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4981 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5041 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5101 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5161 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5221 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5281 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5341 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5401 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5461 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5521 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5581 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5641 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5701 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5761 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5821 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5881 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5941 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6001 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6061 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6121 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6181 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6241 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6301 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6361 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6421 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6481 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6541 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6601 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6661 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6721 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6781 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6841 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6901 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6961 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     7021 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     7081 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     7141 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     7201 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
     7261 gcccgccggg taactcacgg ggtatccatg tccatttctg cggcatccag ccaggatacc
     7321 cgtcctcgct gacgtaatat cccagcgccg caccgctgtc attaatctgc acaccggcac
     7381 ggcagttccg gctgtcgccg gtattgttcg ggttgctgat gcgcttcggg ctgaccatcc
     7441 ggaactgtgt ccggaaaagc cgcgacgaac tggtatccca ggtggcctga acgaacagtt
     7501 caccgttaaa ggcgtgcatg gccacacctt cccgaatcat catggtaaac gtgcgttttc
     7561 gctcaacgtc aatgcagcag cagtcatcct cggcaaactc tttccatgcc gcttcaacct
     7621 cgcgggaaaa ggcacgggct tcttcctccc cgatgcccag atagcgccag cttgggcgat
     7681 gactgagccg gaaaaaagac ccgacgatat gatcctgatg cagctaga
//
