LOCUS       Exported                8047 bp ds-DNA     circular SYN 21-AUG-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8047)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Aug 22, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     UUID: 6043b3f9-53aa-45fa-aa85-d0f3bb13b5fb
            Type: Synthetic
            ConfirmedExperimentally: 0
            CustomMapLabel: PiggyBac-3XHA-EGFP-OMP25
            UseCustomMapLabel: 1
            CreatedBy: Triple Threat
            SequenceClass: UNA
            TransformedInto: Unspecified
FEATURES             Location/Qualifiers
     source          1..8047
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        2..105
                     /gene="bla"
                     /locus_tag="AmpR promoter"
                     /label=AmpR promoter
                     /note="color: #bbbbbb; direction: BOTH"
     promoter        451..469
                     /locus_tag="SP6 promoter"
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
                     /note="color: #bbbbbb; direction: BOTH"
     protein_bind    535..556
                     /locus_tag="CAP binding site"
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator
                     protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /note="color: #31849b; direction: BOTH"
     promoter        571..601
                     /locus_tag="lac promoter"
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="color: #bbbbbb; direction: BOTH"
     protein_bind    609..625
                     /locus_tag="lac operator"
                     /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-I²-D-thiogalactopyranoside (IPTG)."
                     /note="color: #31849b; direction: BOTH"
     primer_bind     633..649
                     /locus_tag="M13 rev"
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     promoter        670..688
                     /locus_tag="T3 promoter"
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #bbbbbb; direction: BOTH"
     enhancer        1225..1604
                     /locus_tag="CMV enhancer"
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #bbbbbb"
     promoter        1607..1882
                     /locus_tag="chicken I²-actin promoter"
                     /label=chicken I²-actin promoter
                     /note="color: #bbbbbb; direction: BOTH"
     intron          1883..2891
                     /locus_tag="chimeric intron"
                     /label=chimeric intron
                     /note="chimera between introns from chicken I²-actin and
                     rabbit I²-globin"
                     /note="color: #444444"
     misc_feature    2949..2972
                     /locus_tag="Flag"
                     /label=Flag
                     /note="color: #00ffff; direction: BOTH"
     CDS             2988..3704
                     /codon_start=1
                     /locus_tag="EGFP"
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="color: #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    3720..4157
                     /locus_tag="OMP25"
                     /label=OMP25
                     /note="color: #ff9900; direction: BOTH"
     misc_feature    4174..4760
                     /locus_tag="IRES2"
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
                     /note="color: #a6acb3; direction: BOTH"
     CDS             4767..5366
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /locus_tag="PuroR"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /note="color: #ccffcc"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    5598..5645
                     /locus_tag="HSV TK poly(A) signal"
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
                     /note="color: #a6acb3"
     promoter        6152..6170
                     /locus_tag="T7 promoter"
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #bbbbbb; direction: BOTH"
     rep_origin      complement(6428..7016)
                     /direction=LEFT
                     /locus_tag="ori"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             7187..8047
                     /codon_start=1
                     /gene="bla"
                     /locus_tag="AmpR"
                     /product="I²-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /note="color: #ccffcc"
                     /translation="MPMLNQ*GTYLSDLSISFIHSCLTPRRVDNYDTGGLTIWPQCCND
                     TARPTLTGSRFISNKPASRKGRAQKWSCNFIRLHPVY*LLPGS*SK*FAS**FAQRCCH
                     CYRHRGVTLVVWYGFIQLRFPTIKASYMIPHVVQKSG*LLRSSDRCQK*VGRSVITHGY
                     GSTA*FSYCHAIRKMLFCDW*VLNQVILRIVYAATELLLPGVNTG*YRAT*QNFKSAHH
                     WKTFFGAKTLKDLTAVEIQFDVTHSCTQLIFSIFYFHQRFWVSKNRKAKCRKKGNKGDT
                     EMLNTH"
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 atgggtggac tatttacggt
     1441 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1501 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1561 ctacttggca gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc
     1621 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
     1681 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg
     1741 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
     1801 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     1861 aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg
     1921 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
     1981 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc
     2041 ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt gcggggggga
     2101 gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct
     2161 gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc
     2221 gaggggagcg cggccggggg cggtgccccg cggtgcgggg gggctgcgag gggaacaaag
     2281 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc
     2341 tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg
     2401 gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg
     2461 ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc
     2521 ggccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     2581 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg
     2641 gaggcgccgc cgcaccccct ctagcgggcg cgggcgaagc ggtgcggcgc cggcaggaag
     2701 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag
     2761 cctcggggct gccgcagggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     2821 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
     2881 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
     2941 aattcatgga ttacaaagac gatgacgata agggtggtgg tggttctatg gtgagcaagg
     3001 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     3061 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     3121 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     3181 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     3241 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     3301 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3361 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3421 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     3481 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     3541 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     3601 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     3661 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggtggt ggtggttcta
     3721 tgaacggaag agtggattat ttggtcactg aggaagagat caatcttacc agagggccct
     3781 cagggctggg cttcaacatc gtcggtggga cagatcagca gtatgtctcc aacgacagtg
     3841 gcatctacgt cagccgcatc aaagaaaatg gggctgcggc cctggatggg cggctccagg
     3901 agggtgataa gatcctttcg gtaaatggcc aagacctaaa gaacctgctg caccaggatg
     3961 ctgtagacct ctttcgtaat gcaggctatg ctgtgtctct gagagtgcag cacaggttac
     4021 aggtgcagaa tggacctata ggacatcgag gtgaagggga cccaagtggt attcccatat
     4081 ttatggtgct ggtgccagtg tttgccctca ccatggtagc agcctgggct ttcatgagat
     4141 accggcaaca actttgaagc ggccgcggat 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 aaaaaccaca 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
//
