LOCUS       sequence-180845-        7077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  production of transgenic animal and recombination using flipase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7077)
  AUTHORS   Lee HJ, Lee HC, Kim YM, Hwang YS, Park YH, Park TS, Han JY
  TITLE     Site-specific recombination in the chicken genome using Flipase 
            recombinase-mediated cassette exchange.
  JOURNAL   FASEB J. 2016 Feb;30(2):555-63. doi: 10.1096/fj.15-274712. Epub 2015
            Oct 6.
  PUBMED    26443821
REFERENCE   2  (bases 1 to 7077)
  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: 
            "10.1096/fj.15-274712"; journalName: "FASEB J"; date: "2016-02"; 
            volume: "30"; issue: "2"; pages: "555-63"
COMMENT     Sequence Label: piggyBac CMV-GFP-FRT
FEATURES             Location/Qualifiers
     source          1..7077
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      686..695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(692..694,695..697,698..1411)
                     /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(692..694,695..697,698..1408)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(737..758)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(998..1017)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1345..1366
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1439..1457)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1440..1457)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1477..1494)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1483..1707
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1717..1764
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     repeat_region   2114..2176
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(2243..2261)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2268..2285)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2268..2284)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2277..2299)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2426..2881
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2513..2532)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2723..2744
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2907..3011
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3012..3080,3081..3872)
                     /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(3230..3249)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4043..4631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4532..4551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4785..4802
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4919..4940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4955..4960,4961..4978,4979..4985)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4993..5009
                     /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     4998..5020
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5017..5033
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5017..5033
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5054..5072
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   5221..5255
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    5544..5577
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     protein_bind    complement(5598..5631)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             5916..6719
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5979..5998)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6589..6608
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6726..6774
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    complement(6785..6818)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6887..6906)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac
      721 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      781 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
      841 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
      901 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      961 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1021 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1081 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1141 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1201 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1261 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1321 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1381 cactctcggc atggacgagc tgtacaagta aagcggccgc gactctagag ggccctattc
     1441 tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     1501 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     1561 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     1621 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     1681 atgctgggga tgcggtgggc tctatggctt gtcgacgaag ttcctattcc gaagttccta
     1741 ttctctagaa agtataggaa cttcctcgag ttaattaacg agagcataat attgatatgt
     1801 gccaaagttg tttctgactg actaataagt ataatttgtt tctattatgt ataggttaag
     1861 ctaattactt attttataat acaacatgac tgtttttaaa gtacaaaata agtttatttt
     1921 tgtaaaagag agaatgttta aaagttttgt tactttatag aagaaatttt gagtttttgt
     1981 ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt
     2041 aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca
     2101 gaccgataaa acacatgcgt caattttacg catgattatc tttaacgtac gtcacaatat
     2161 gattatcttt ctagggttaa ataatagttt ctaatttttt tattattcag cctgctgtcg
     2221 tgaataccga gctccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
     2281 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2341 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2401 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2461 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2521 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2581 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2641 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2701 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2761 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2821 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     2881 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2941 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3001 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3061 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3121 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3181 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3241 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3301 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3361 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3421 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3481 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3541 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3601 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3661 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3721 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3781 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3841 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3901 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3961 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4021 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4081 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4141 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4201 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4261 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4321 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4381 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4441 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4501 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4561 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4621 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4681 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4741 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4801 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     4861 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     4921 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     4981 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     5041 acgccaagct cgaaattaac cctcactaaa gggaacaaaa gctggtacct cgcgcgactt
     5101 ggtttgccat tctttagcgc gcgtcgcgtc acacagcttg gccacaatgt ggtttttgtc
     5161 aaacgaagat tctatgacgt gtttaaagtt taggtcgagt aaagcgcaaa tcttttttaa
     5221 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt
     5281 tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc
     5341 ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt
     5401 gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact catacgataa
     5461 ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt
     5521 atagatatcg aagttcctat tccgaagttc ctattcttca aatagtatag gaacttcgtc
     5581 gagatcgatg gcgcgccata acttcgtata gcatacatta tacgaagtta tgaatttcga
     5641 gcagtgtggt tttcaagagg aagcaaaaag cctctccacc caggcctgga atgtttccac
     5701 ccaatgtcga gcagtgtggt tttgcaagag gaagcaaaaa gcctctccac ccaggcctgg
     5761 aatgtttcca cccaatgtcg agcaaacccc gcccagcgtc ttgtcattgg cgaattcgaa
     5821 cacgcagatg cagtcggggc ggcgcggtcc caggtccact tcgcatatta aggtgacgcg
     5881 tgtggcctcg aacaccgagc gaccctgcag ccaatatggg atcggccatt gaacaagatg
     5941 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     6001 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     6061 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     6121 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     6181 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     6241 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     6301 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     6361 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     6421 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg
     6481 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     6541 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     6601 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     6661 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     6721 gggatcggca ataaaaagac agaataaaac gcacgggtgt tgggtcgttt gttcggatca
     6781 attcataact tcgtatagca tacattatac gaagttatgg cgcgccttaa ttaaggatcc
     6841 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     6901 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     6961 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     7021 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
