LOCUS       sequence-135606-        5901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  +36GFP-LPETG for expression in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5901)
  AUTHORS   Li M, Tao Y, Shu Y, LaRochelle JR, Steinauer A, Thompson D, 
            Schepartz A, Chen ZY, Liu DR
  TITLE     Discovery and Characterization of a Peptide That Enhances Endosomal 
            Escape of Delivered Proteins in Vitro and in Vivo.
  JOURNAL   J Am Chem Soc. 2015 Nov 11;137(44):14084-93. doi: 
            10.1021/jacs.5b05694. Epub 2015 Oct 30.
  PUBMED    26465072
REFERENCE   2  (bases 1 to 5901)
  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: 
            "10.1021/jacs.5b05694"; journalName: "J Am Chem Soc"; date: 
            "2015-11-11- 11"; volume: "137"; issue: "44"; pages: "14084-93"
COMMENT     Sequence Label: +36GFP-LPETG
FEATURES             Location/Qualifiers
     source          1..5901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..26
                     /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)."
     RBS             41..63
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             811..828
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             847..891
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(929..947)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      943..990
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1027..1482
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1114..1133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1324..1345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1597..2388,2389..2457))
                     /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     2220..2239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2458..2550)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2631..3219
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3120..3139
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3373..3390
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3405..3547
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3473..3492)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3633..3655
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3649..3840)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4412..5494)
                     /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     5456..5475
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5495..5572)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5778..5797
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5885..5901,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5885..5901,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
       61 agatatcata tgagcaaagg tgaacgtctg tttcgtggta aagtaccgat cttagtggaa
      121 ttaaagggcg acgtgaacgg tcataaattt agcgtgcgcg gcaaaggcaa aggtgacgct
      181 acccgtggta aattgaccct gaagtttatt tgcacaacag gcaaattacc cgttccgtgg
      241 cccaccttag tgaccaccct gacctatggc gttcagtgct tcagtcgtta ccctaaacac
      301 atgaaacgtc acgatttttt caaatcagcc atgcctaaag gatatgttca agagcgtaca
      361 atcagcttca agaaggatgg caaatataaa acgcgtgcgg aagtgaaatt tgaaggccgc
      421 acattagtaa atcgtatcaa actgaaaggt cgtgacttca aagaaaaagg caacatttta
      481 ggccataaac tgcgttataa ctttaattct cataaggtgt atattacggc cgataaacgc
      541 aagaatggta tcaaggcaaa attcaaaatt cgccataacg tgaaagacgg cagcgttcaa
      601 ttagcggatc attatcaaca aaacacgccg attggtcgcg ggcctgtact gttacctcgc
      661 aaccactacc tgagcacccg ttctaaactg agcaaagatc cgaaagaaaa acgcgatcac
      721 atggttctgt tagaattcgt gaccgctgca ggcattaagc acggacgcga cgaacgctac
      781 aagaccggtg gtagcctccc ggaaaccggt catcaccacc accatcacta atgactcgag
      841 tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc
      901 gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
      961 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1021 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1081 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1141 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1201 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1261 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1321 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1381 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1441 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat
     1501 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1561 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1621 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1681 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1741 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1801 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1861 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     1921 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     1981 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2041 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2101 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2161 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2221 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2281 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2341 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2401 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2461 cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2521 tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag
     2581 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2641 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2701 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2761 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2821 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2881 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2941 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3001 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3061 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3121 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3181 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3241 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3301 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3361 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3421 ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct
     3481 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     3541 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3601 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3661 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     3721 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     3781 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     3841 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     3901 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     3961 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4021 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4081 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4141 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4201 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4261 gcctagccgg gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg
     4321 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     4381 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4441 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4501 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     4561 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     4621 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     4681 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     4741 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     4801 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     4861 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     4921 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     4981 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5041 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5101 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5161 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5221 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5281 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     5341 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     5401 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     5461 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     5521 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     5581 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     5641 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     5701 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     5761 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     5821 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     5881 aaattaatac gactcactat a
//
