LOCUS       sequence-487556-        8047 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  Expression of OMP25-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8047)
  TITLE     Hengbin lab plasmids 2025
REFERENCE   2  (bases 1 to 8047)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Piggybac-omp25-GFP
FEATURES             Location/Qualifiers
     source          1..8047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     173..191
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(229..251)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     351..370
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        451..469
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     451..468
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    535..556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(571..576,577..594,595..601)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    609..625
                     /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     614..636
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     633..649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     633..649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        670..688
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   871..933
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1225..1604
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1884..2900
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2824..2846
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2908..2927
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2958..2981
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2997..2999,3000..3002,3003..3713)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2997..2999,3000..3002,3003..3713)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3042..3063)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3303..3322)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3650..3671
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(4174..4748,4749..4751,4752..4760)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4341..4358)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4568..4587
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4767..5366
                     /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(4767..4786)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5263..5283
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5554..5573)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5598..5645
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   5993..6027
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6151..6170)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6152..6170)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6257..6274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6428..7016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6508..6527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7187..7978,7979..8047))
                     /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     7810..7829
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      361 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
      421 atacataacc ttatgtatca tacacatacg atttaggtga cactatagaa ctcgacctcg
      481 aggctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      541 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      601 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      661 caagctcgaa attaaccctc actaaaggga acaaaagctg gagctcgtct ttgatcaaaa
      721 cgcaaatcga cgaaaatgtg tcggacaata tcaagtcgat gagcgaaaaa ctaaaaaggc
      781 tagaatacga caatctcaca gacagcgttg agatatacgg tattcacgac agcaggctga
      841 ataataaaaa aattagaaac tattatttaa ccctagaaag ataatcatat tgtgacgtac
      901 gttaaagata atcatgcgta aaattgacgc atgtgtttta tcgatctgta tatcgaggtt
      961 tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata
     1021 aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa
     1081 tttcttctat aaagtaacaa aacttttaaa cattctctct tttacaaaaa taaacttatt
     1141 ttgtacttta aaaacagtca tgttgtatta taaaataagt aattagctta acttatacat
     1201 aatagaaaca aattatactt attagacatt gattattgac tagttattaa tagtaatcaa
     1261 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1321 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1381 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1441 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1501 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1561 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     1621 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     1681 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggggg cgccaggggg
     1741 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggcgcccc
     1801 gggggggggg gggcgcgggg ggtccttttt tgcggggggg gggggggggg gggcgcccta
     1861 aaaaaagcgg ggggcggggg gggggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     1921 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1981 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
     2041 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
     2101 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
     2161 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     2221 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     2281 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     2341 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     2401 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc
     2461 aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc
     2521 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     2581 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     2641 aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg
     2701 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc
     2761 ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg
     2821 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     2881 ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt
     2941 ttggcaaaga attcatggat tacaaagacg atgacgataa gggtggtggt ggttctatgg
     3001 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     3061 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     3121 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     3181 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     3241 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     3301 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     3361 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     3421 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     3481 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     3541 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     3601 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     3661 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggtggtg
     3721 gtggttctat gaacggaaga gtggattatt tggtcactga ggaagagatc aatcttacca
     3781 gagggccctc agggctgggc ttcaacatcg tcggtgggac agatcagcag tatgtctcca
     3841 acgacagtgg catctacgtc agccgcatca aagaaaatgg ggctgcggcc ctggatgggc
     3901 ggctccagga gggtgataag atcctttcgg taaatggcca agacctaaag aacctgctgc
     3961 accaggatgc tgtagacctc tttcgtaatg caggctatgc tgtgtctctg agagtgcagc
     4021 acaggttaca ggtgcagaat ggacctatag gacatcgagg tgaaggggac ccaagtggta
     4081 ttcccatatt tatggtgctg gtgccagtgt ttgccctcac catggtagca gcctgggctt
     4141 tcatgagata ccggcaacaa ctttgaggat ccgcccctct ccctcccccc cccctaacgt
     4201 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
     4261 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag
     4321 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa
     4381 ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag
     4441 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga
     4501 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
     4561 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc
     4621 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag
     4681 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg
     4741 atgataatat ggccacaacc atgggaatga ccgagtacaa gcccacggtg cgcctcgcca
     4801 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     4861 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     4921 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     4981 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     5041 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     5101 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     5161 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     5221 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     5281 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     5341 gcatgacccg caagcccggt gcctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     5401 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5461 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5521 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     5581 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     5641 tgttcggcgc gccatcgaag cagctccagc ctacacaatc gctcaagacg tgtaatgctt
     5701 ttattatata ttagtcacga tatctataac aagaaaatat atatataata agttatcacg
     5761 taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta
     5821 aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact
     5881 taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg
     5941 cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc
     6001 aattttacgc agactatctt tctagggtta aaaaagattt gcgctttact cgacctaaac
     6061 tttaaacacg tcatagaatc ttcgtttgac aaaaaccaaa ttgtggggta ccgagctcgt
     6121 cgacatcgat gatatcagat ctgccggtct ccctatagtg agtcgtatta atttcgataa
     6181 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6241 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6301 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6361 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6421 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6481 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6541 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6601 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6661 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6721 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6781 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6841 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6901 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6961 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     7021 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     7081 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     7141 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     7201 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     7261 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7321 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7381 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7441 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7501 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7561 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7621 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7681 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7741 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7801 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7861 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7921 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7981 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     8041 tactcat
//
