LOCUS       sequence-314239-        6146 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresion of the fusion protein of the lambda-N domain of the lambda
            phage and ampR gene (encoding the enzyme beta-lactamase without the 
            native signal sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6146)
  AUTHORS   Moya Ramirez I, Kontoravdi C, Polizzi KM
  TITLE     Low-cost and user-friendly biosensor to test the integrity of mRNA 
            molecules suitable for field applications.
  JOURNAL   Biosens Bioelectron. 2019 Jul 15;137:199-206. doi: 
            10.1016/j.bios.2019.05.008. Epub 2019 May 7.
  PUBMED    31100599
REFERENCE   2  (bases 1 to 6146)
  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"; journalName: "Biosens 
            Bioelectron."; date: "2019-07-15"; volume: "137:199-206. doi"; 
            pages: " 10.1016/j.bios.2019.05.008. Epub 2019 May 7"
COMMENT     Sequence Label: pET28a-lambda-N-beta-Lac
FEATURES             Location/Qualifiers
     source          1..6146
                     /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             221..1006
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /label=bla(M)
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST
                     FKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
                     DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATT
                     LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA
                     LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     primer_bind     complement(367..386)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             1007..1024
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1077..1095)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1091..1138
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1175..1630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1262..1281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1472..1493
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1723..2538)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1723..2538)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2446..2465
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2530..2549
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2660..3248
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3149..3168
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3402..3419
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3434..3576
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3502..3521)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3662..3684
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3678..3869)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4678..5760)
                     /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     5722..5741
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5761..5838)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6044..6063
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccaat ggggaatgca aaaacacgcc gccgcgaacg
      121 tcgcgcagag aaacaggctc aatggaaagc agcaaatggt gcgggggccg gcgcaggtgc
      181 aggcgctggt gcaggcgcgg gtgccggcgc gggtgctcac ccagaaacgc tggtgaaagt
      241 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      301 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      361 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      421 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      481 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      541 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      601 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      661 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      721 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      781 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      841 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      901 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
      961 aaatagacag atcgctgaga taggtgcctc actgattaag cattggcacc accaccacca
     1021 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1081 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1141 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1201 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1261 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1321 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1381 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1441 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1501 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1561 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1621 tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1681 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     1741 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     1801 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     1861 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     1921 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     1981 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     2041 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     2101 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     2161 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     2221 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     2281 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     2341 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     2401 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     2461 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     2521 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     2581 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2641 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2701 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2761 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2821 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2881 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2941 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3001 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3061 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3121 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3181 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3241 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3301 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3361 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3421 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3481 gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3541 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3601 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3661 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3721 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3781 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3841 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3901 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3961 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4021 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4081 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4141 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4201 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4261 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4321 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     4381 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     4441 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     4501 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     4561 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     4621 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4681 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4741 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4801 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4861 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4921 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     4981 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     5041 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     5101 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     5161 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     5221 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5281 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     5341 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     5401 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     5461 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     5521 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     5581 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5641 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5701 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5761 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5821 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5881 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5941 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     6001 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     6061 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     6121 ggatcgagat ctcgatcccg cgaaat
//
