LOCUS       sequence-176142-        5893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP with +9 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-(pos9)GFP
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..67
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    82..221
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(147..166)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     307..329
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(323..514)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1086..2168)
                     /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     2130..2149
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2169..2246)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2452..2471
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2559..2578
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2559..2577
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2578..2602
                     /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             2617..2639
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2652..2669
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3417..3461
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3499..3517)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3513..3560
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3597..4052
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3684..3703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3894..3915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4167..4958,4959..5027))
                     /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     4790..4809
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5028..5120)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5201..5789
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5690..5709
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
       61 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
      121 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
      181 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
      241 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
      301 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
      361 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
      421 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
      481 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
      541 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
      601 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
      661 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
      721 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
      781 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
      841 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
      901 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
      961 gcacgatcat gctagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     1021 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     1081 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1141 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     1201 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     1261 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     1321 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     1381 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     1441 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     1501 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     1561 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     1621 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     1681 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     1741 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     1801 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     1861 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     1921 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     1981 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     2041 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     2101 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     2161 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     2221 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     2281 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     2341 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     2401 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     2461 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     2521 ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta atacgactca ctatagggga
     2581 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     2641 ataccatggg tcatcaccac caccatcacg gtggcgctag caaaggtgaa gagctgttta
     2701 cgggtgtagt accgatctta gtggaattag acggcgacgt gaacggtcac aaatttagcg
     2761 tgcgcggcga aggcgaaggt gacgctacca atggtaaatt gaccctgaag tttatttgca
     2821 caacaggcaa attacccgtt ccgtggccca ccttagtgac caccctgacc tatggcgttc
     2881 agtgcttcag tcgttaccca gatcatatga aacgtcacga ttttttcaaa tcagccatgc
     2941 ctaaaggata tgttcaagag cgtacaatca gcttcaagaa ggatggcaaa tataaaacgc
     3001 gtgcggaagt gaaatttgaa ggccgcacat tagtaaatcg tatcaaactg aaaggtcgtg
     3061 acttcaaaga aaaaggcaac attttaggcc ataaactgcg ttataacttt aattctcata
     3121 aggtgtatat tacggccgat aaacagaaaa acggtatcaa ggcaaatttc aaaattcgcc
     3181 ataacgtgga agacggcagc gttcaattag cggatcatta tcaacaaaac acgccgattg
     3241 gtgacgggcc tgtactgtta cctgacaacc actacctgag cacccagtca gcactgagca
     3301 aagatccgaa cgaaaaacgc gatcacatgg ttctgttaga attcgtgacc gctgcaggca
     3361 ttactcacgg aatggacgaa ctctacaagt aataatgagg taccctcgag tctggtaaag
     3421 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     3481 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     3541 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     3601 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3661 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3721 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3781 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3841 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3901 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3961 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4021 gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca
     4081 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4141 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4201 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4261 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4321 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4381 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4441 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4501 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4561 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4621 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4681 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4741 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4801 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4861 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4921 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4981 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5041 caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5101 tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5161 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5221 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5281 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5341 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5401 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5461 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5521 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5581 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5641 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5701 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5761 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5821 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5881 ataaccgtat tac
//
