LOCUS       sequence-130226-        6036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U_+36GFP-Cys fusion expression in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6036)
  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 6036)
  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: U_(pos36GFP)-Cys
FEATURES             Location/Qualifiers
     source          1..6036
                     /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             946..963
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             982..1026
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1064..1082)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1078..1125
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1162..1617
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1249..1268)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1459..1480
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1732..2523,2524..2592))
                     /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     2355..2374
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2593..2685)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2766..3354
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3255..3274
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3508..3525
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3540..3682
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3608..3627)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3768..3790
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3784..3975)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4547..5629)
                     /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     5591..5610
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5630..5707)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5913..5932
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6020..6036,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6020..6036,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
       61 agatatcata tgggcctgtt tgatattatt aaaaaagtgg cgagcgtgat tggcggcctg
      121 gctagcggtg gtagcggtgg ttctggtggt tctggtggta gcggcggtag cggtggtagc
      181 ggtggtagcg gtggcagcgg cggttctagc aaaggtgaac gtctgtttcg tggtaaagta
      241 ccgatcttag tggaattaaa gggcgacgtg aacggtcata aatttagcgt gcgcggcaaa
      301 ggcaaaggtg acgctacccg tggtaaattg accctgaagt ttatttgcac aacaggcaaa
      361 ttacccgttc cgtggcccac cttagtgacc accctgacct atggcgttca gtgcttcagt
      421 cgttacccta aacacatgaa acgtcacgat tttttcaaat cagccatgcc taaaggatat
      481 gttcaagagc gtacaatcag cttcaagaag gatggcaaat ataaaacgcg tgcggaagtg
      541 aaatttgaag gccgcacatt agtaaatcgt atcaaactga aaggtcgtga cttcaaagaa
      601 aaaggcaaca ttttaggcca taaactgcgt tataacttta attctcataa ggtgtatatt
      661 acggccgata aacgcaagaa tggtatcaag gcaaaattca aaattcgcca taacgtgaaa
      721 gacggcagcg ttcaattagc ggatcattat caacaaaaca cgccgattgg tcgcgggcct
      781 gtactgttac ctcgcaacca ctacctgagc acccgttcta aacttagcaa agatccgaaa
      841 gaaaaacgcg atcacatggt tctgttagaa ttcgtgaccg ctgcaggcat taagcacgga
      901 cgcgacgaac gctacaagac cggtggtagc ggatgcggag gatcccatca ccaccaccat
      961 cactaatgac tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg
     1021 gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta
     1081 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1141 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1201 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1261 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1321 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1381 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1441 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1501 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1561 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg
     1621 gcggcacgat ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1681 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1741 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     1801 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     1861 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     1921 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     1981 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2041 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2101 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2161 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2221 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2281 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2341 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2401 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2461 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2521 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2581 ttgaatactc atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc
     2641 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa
     2701 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2761 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2821 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2881 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2941 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3001 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3061 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3121 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3181 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3241 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3301 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3361 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3421 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3481 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3541 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca
     3601 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3661 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3721 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3781 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3841 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3901 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3961 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4021 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4081 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4141 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4201 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4261 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4321 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4381 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat
     4441 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4501 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4561 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4621 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     4681 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     4741 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     4801 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     4861 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4921 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     4981 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     5041 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     5101 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     5161 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5221 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5281 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5341 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     5401 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     5461 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     5521 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     5581 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     5641 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     5701 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     5761 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     5821 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     5881 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     5941 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     6001 gatctcgatc ccgcgaaatt aatacgactc actata
//
