LOCUS       sequence-485904-        6268 bp    DNA     circular SYN 12-SEP-2025
DEFINITION  Expresses eGFP fluorophore with fused p2c targeting ligand for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6268)
  AUTHORS   Chaverra-Rodriguez D, Macias VM, Hughes GL, Pujhari S, Suzuki Y, 
            Peterson DR, Kim D, McKeand S, Rasgon JL
  TITLE     Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod 
            ovaries for heritable germline gene editing.
  JOURNAL   Nat Commun. 2018 Aug 1;9(1):3008. doi: 10.1038/s41467-018-05425-9.
  PUBMED    30068905
REFERENCE   2  (bases 1 to 6268)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 12, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-08-1"; volume: "9(1):3008. doi"; pages: " 
            10.1038/s41467-018-05425-9"
COMMENT     Sequence Label: pET28(a)-p2c-eGFP
FEATURES             Location/Qualifiers
     source          1..6268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             42..59
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             69..86
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             90..122
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(315..317,318..320,321..1028)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKGD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(360..381)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     968..989
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1075..1092
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1145..1163)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1159..1206
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1243..1698
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1330..1349)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1540..1561
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1790..2605)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1790..2605)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2513..2532
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2597..2616
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2727..3315
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3216..3235
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3469..3486
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3501..3640
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3566..3585)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3726..3748
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3742..3933)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4742..5824)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5786..5805
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5825..5902)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6108..6127
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6211..6230
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6211..6229
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6230..6254
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
       61 gcagcggcct ggtgccgcgc ggcagccata tggctagcat gactggtgga cagcaaatgg
      121 gtcgcggatc caatctgcag cagcagcgcc agcacggcaa gaacggcaac caggactacc
      181 aggatcagag caacgaacag aggaagaacc agaggaccag cagcgaggag gactacagcg
      241 aggaggttaa gaacgagctc aggcgcgccg gaggaggcgg cagcggtggt ggaggaagcg
      301 gtggaggcgg gagcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      361 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      421 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      481 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      541 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      601 agcgcaccat cttcttcaag ggcgacggca actacaagac ccgcgccgag gtgaagttcg
      661 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      721 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
      781 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
      841 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      901 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
      961 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1021 agctgtacgg atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac
     1081 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     1141 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1201 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     1261 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1321 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1381 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1441 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1501 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1561 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1621 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1681 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1741 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     1801 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     1861 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1921 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1981 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     2041 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2101 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2161 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2221 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2281 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     2341 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     2401 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     2461 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     2521 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     2581 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2641 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2701 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2761 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagccacca
     2821 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     2881 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2941 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3001 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3061 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3121 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3181 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3241 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3301 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3361 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3421 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3481 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3541 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3601 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3661 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3721 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3781 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     3841 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     3901 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     3961 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4021 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4081 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4141 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4201 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4261 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4321 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4381 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     4441 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4501 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4561 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4621 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4681 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4741 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4801 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4861 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4921 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4981 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     5041 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5101 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5161 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5221 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5281 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5341 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5401 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5461 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     5521 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5581 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5641 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5701 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5761 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5821 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5881 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     5941 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     6001 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     6061 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     6121 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     6181 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     6241 cggataacaa ttcccctcta gaaataat
//
