LOCUS       sequence-431124-        6513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Beta-Lactamase protein in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6513)
  AUTHORS   D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H, Prasad V,
            Lebbink RJ, Rehmann H, Geijsen N
  TITLE     Efficient Intracellular Delivery of Native Proteins.
  JOURNAL   Cell. 2015 Apr 23;161(3):674-690. doi: 10.1016/j.cell.2015.03.028.
  PUBMED    25910214
REFERENCE   2  (bases 1 to 6513)
  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.cell.2015.03"; journalName: "Cell"; date: "2015-04-23- 
            23"; volume: "161"; issue: "3"; pages: "674-690"
COMMENT     Sequence Label: Beta-Lactamase
FEATURES             Location/Qualifiers
     source          1..6513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             169..963
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /label=bla(M)
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="MHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMM
                     STFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAIT
                     MSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMA
                     TTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGII
                     AALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     primer_bind     complement(1014..1032)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1028..1075
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1250..1255,1256..1272,1273..1278))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1305..1323)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1391..1495
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1496..1564,1565..2356)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2527..3115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3016..3035
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3269..3286
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3301..3440
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3366..3385)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3526..3548
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3542..3733)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5045..6127)
                     /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     6089..6108
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6128..6205)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6411..6430
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggctcatcgc atcatcatca tcatcattca
      121 tcgggtctgg ttccgcgcgg ttcacacggt ggtggttcgg cggccgccat gcacccagaa
      181 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
      241 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
      301 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
      361 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
      421 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
      481 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
      541 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
      601 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
      661 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
      721 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
      781 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
      841 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
      901 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
      961 taaggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1021 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1081 aggaggaact atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct
     1141 atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc
     1201 atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta
     1261 tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct
     1321 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1381 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1441 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1501 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1561 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1621 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1681 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     1741 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1801 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1861 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     1921 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     1981 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2041 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2101 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2161 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2221 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2281 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2341 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2401 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2461 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2521 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2581 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2641 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2701 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2761 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2821 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2881 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2941 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3001 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3061 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3121 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3181 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3241 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3301 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     3361 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     3421 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3481 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3541 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     3601 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     3661 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     3721 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     3781 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     3841 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     3901 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     3961 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4021 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     4081 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     4141 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     4201 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
     4261 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
     4321 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     4381 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     4441 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
     4501 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
     4561 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
     4621 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     4681 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     4741 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4801 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4861 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4921 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4981 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5041 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5101 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5161 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5221 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5281 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5341 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5401 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5461 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5521 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5581 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5641 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5701 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5761 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5821 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5881 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5941 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6001 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6061 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6121 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6181 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6241 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6301 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6361 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6421 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6481 gcgtagagga tcgagatctc gatcccgcga aat
//
