LOCUS       sequence-176147-        5893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  [Edit] Expresses GFP with +15 theoretical charge in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5893)
  AUTHORS   Thompson DB, Villasenor R, Dorr BM, Zerial M, Liu DR
  TITLE     Cellular uptake mechanisms and endosomal trafficking of supercharged
            proteins.
  JOURNAL   Chem Biol. 2012 Jul 27;19(7):831-43. doi: 
            10.1016/j.chembiol.2012.06.014.
  PUBMED    22840771
REFERENCE   2  (bases 1 to 5893)
  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: 
            "10.1016/j.chembiol.2012.06"; journalName: "Chem Biol"; date: 
            "2012-07-27- 27"; volume: "19"; issue: "7"; pages: "831-43"
COMMENT     Sequence Label: pET-6xHis-(pos15)GFP
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(321..338)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             351..373
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    388..412
                     /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     complement(412..431)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(413..431)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(519..538)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        744..821
                     /gene="lacI"
                     /label=lacI promoter
     CDS             822..1904
                     /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     complement(841..860)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2476..2667
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2661..2683)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2769..2908
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2824..2843
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2923..2940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3094..3682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3174..3193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3763..3855
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3856..3924,3925..4716)
                     /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     complement(4074..4093)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4831..5286)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4968..4989)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5180..5199
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5323..5370)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5366..5384
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5422..5466)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
ORIGIN
        1 gtgccatcct tcttgaagct gattgtacgc tcttgaacat atccttcagg catggctgat
       61 ttgaaaaaat cgtgacgttt catatgttta gggtaacgac tgaagcactg aacgccatag
      121 gtcagggtgg tcactaaggt gggccacgga acgggtaatt tgcctgttgt gcaaataaac
      181 ttcagggtca atttaccacg ggtagcgtca ccttcgcctt cgccgcgcac gctaaatttg
      241 tgaccgttca cgtcgccgtc taattccact aagatcggta ctacacccgt aaacagacgt
      301 tcacctttgc tagcgccacc gtgatggtgg tggtgatgac ccatggtata tctccttctt
      361 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt
      421 gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc ctctacgccg gacgcatcgt
      481 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
      541 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
      601 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
      661 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
      721 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
      781 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
      841 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
      901 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
      961 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     1021 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     1081 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     1141 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     1201 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     1261 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     1321 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     1381 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     1441 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     1501 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     1561 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     1621 acatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     1681 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     1741 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     1801 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     1861 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     1921 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     1981 ccagtcagct ccttccggtg ggcgcggggc atgactagca tgatcgtgct cctgtcgttg
     2041 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
     2101 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
     2161 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt
     2221 atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta
     2281 ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc
     2341 gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt
     2401 atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct
     2461 tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc
     2521 agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca
     2581 gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt
     2641 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     2701 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     2761 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     2821 atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag
     2881 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     2941 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3001 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3061 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3121 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3181 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3241 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3301 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3361 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3421 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3481 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     3541 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3601 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3661 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3721 gtggaacgaa aactcacgtt aagggatttt ggtcatgacc tatttgttta tttttctaaa
     3781 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caatcatgat
     3841 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3901 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3961 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4021 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4081 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4141 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4201 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4261 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4321 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4381 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4441 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4501 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4561 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4621 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4681 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4741 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4801 tttgataatc tcatgccatc gtgccgccag aaattgtaaa cgttaatatt ttgttaaaat
     4861 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     4921 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     4981 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     5041 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     5101 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     5161 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     5221 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     5281 gcgcgtccca ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac
     5341 ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag cagcctaggt
     5401 taattaagct gcgctagtag acgagtccat gtgctggcgt tcaaatttcg cagcagcggt
     5461 ttctttacca gactcgaggg tacctcatta ttacttgtag agttcgtcca ttccgtgagt
     5521 aatgcctgca gcggtcacga attctaacag aaccatgtga tcgcgttttt ctttcggatc
     5581 tttgctcagt gctgaacggg tgctcaggta gtggttgcga ggtaacagta caggcccgcg
     5641 accaatcggc gtgttttgtt gataatgatc cgctaattga acgctgccgt ctttcacgtt
     5701 atggcgaatt ttgaaatttg ccttgatacc attcttgcgt ttatcggccg taatatacac
     5761 gttatgagaa ttaaagttat attccagctt atggcctaaa atgttgcctt tttctttgaa
     5821 gtcacgacct ttcagttcga tacggtttac taatgtgcgg ccttcaaatt tcacttccgc
     5881 acgcgtttta tag
//
