LOCUS       sequence-324289-        7119 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible Perfringolysin O (PFO) expression and purification, 
            controlled under hybrid IPTG-regulated T7/LacO promoter (pRT30).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7119)
  AUTHORS   Toparlak OD, Zasso J, Bridi S, Serra MD, Macchi P, Conti L, Baudet 
            ML, Mansy SS
  TITLE     Artificial cells drive neural differentiation.
  JOURNAL   Sci Adv. 2020 Sep 18;6(38). pii: 6/38/eabb4920. doi: 
            10.1126/sciadv.abb4920. Print 2020 Sep.
  PUBMED    32948587
REFERENCE   2  (bases 1 to 7119)
  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: "Sci Adv."; 
            date: "2020-09-18"; volume: "6(38). pii: 6/38/eabb4920. doi"; pages:
            " 10.1126/sciadv.abb4920. Print 2020 Sep"
COMMENT     Sequence Label: DT044A
FEATURES             Location/Qualifiers
     source          1..7119
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(16..21,22..38,39..44)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(219..266)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     262..280
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1756..1773)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1783..1800)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1819..1841
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1856..1880
                     /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(1880..1899)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1881..1899)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1983..2002)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2208..2285
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2286..3368
                     /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(2305..2324)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4680..4871
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4865..4887)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4973..5112
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5028..5047
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5127..5144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5298..5886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5378..5397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6057..6848,6849..6917))
                     /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"
     primer_bind     6680..6699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6918..7022)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7090..7108
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 caagaattct catgtttgac agcttatcat cgataagctt taatgcggta gtttatcaca
       61 gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat
      121 cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg tactgccggg
      181 cctcttgcgg gatatccgga tatagttcct cctttcagca aaaaacccct caagacccgt
      241 ttagaggccc caaggggtta tgctagttat tgctcagcgg tggcagcagc caactcagct
      301 tcctttcggg ctttgttagc agccggatcc tcaattatag gtaatgcttg aacccggata
      361 cagggtggtc ccccaaatcg aaacgttgat attattcgtc agcggcacat catattcgct
      421 aattacatca cgccaccatt cccaggccag ccccgttgct tcacgtgctt taatgcggat
      481 gttgcgcgca ttggcttcca gcgggataac ggtgctatag tgggcagttt tgtcctggta
      541 attgccgtcc caagttttat gcgtcagaac ttcgttacct tctttatcgt atgagacctc
      601 gtcccacgcc acttcaaact gagcgacata cgcaccggaa tgatccaggt tgattttacc
      661 tttagaatat tcggtactgg tggtttcaat gtaatcggtc ttgttatgga cggcggcaac
      721 gctgttgtct ttcaggaaga cagaggtata gctgattgga taagccggat tcttcgtact
      781 aaaagtggca ttatctttga tgactttgcg gatttcgtca aaatctttcg tcaccacttt
      841 attatgttcc tgtgcatccc ctcccagcac aactgctgtg aaactgctat tttcataaat
      901 atctttatac tgttgactat ttttaatatc tgtatttttg atcagagctt taaaagcagc
      961 ttggacatct ttcgaggagc tagtggtttc cagtttaaca taaatggtgc gaccataggc
     1021 aacattagaa accatcagcg gcggagcttc gttgctaaca cctttttgct tcagatcatt
     1081 aaaagtaacg ctatcatcga acagatcaga cggatttttc ggcagatcgg cactgactgt
     1141 ataaaagatc tgcttatatg ccaggatcat tacttttttc tcgttatttg ctacggcgtt
     1201 gaagtcaaca cccagcgaat tttccagcac tttagcgttc acattcaggg ccgatgagat
     1261 ttgggattta ctatacacca tggattcgga gtactgggtg cgcgccggca gggtgtgggt
     1321 gctgctatat ttttcattcc atttgctaac cagttcatca atggcgccgc tgactttacc
     1381 ataagttgga tcatccacct tgatagagtt ttcacctttc agacccggca gatcaatgtt
     1441 gatattgatc ggtttacgtt ttaccatcag aatagtaggg cgattttcga caaatgcttt
     1501 atcagccagt tgcagcgcac caggatacgt gcgatcatta acactgtcaa taatggaaat
     1561 atcaaccggt gaagtagtca ggctacgttt ctggcgttcg actacaatga atttattgcc
     1621 cgctttttta ccctctttag gaacaaacga ttcaatttta tcaccattgc tggccaggac
     1681 ttcattgcgg ttatagctca gcgaactgat gccactatca atcgattggt ttttatccgt
     1741 gatatctttc atatggctgc cgcgcggcac caggccgctg ctgtgatgat gatgatgatg
     1801 gctgctgccc atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     1861 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
     1921 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     1981 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     2041 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     2101 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     2161 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     2221 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     2281 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     2341 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     2401 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     2461 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     2521 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     2581 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     2641 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     2701 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     2761 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     2821 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     2881 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     2941 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     3001 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     3061 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     3121 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     3181 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     3241 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     3301 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3361 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     3421 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     3481 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     3541 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     3601 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     3661 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc
     3721 tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc
     3781 gcttccggcg gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac
     3841 gaccatcagg gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcatt
     3901 ggaccgctga tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca
     3961 tggattgtag gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg
     4021 agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc
     4081 caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc
     4141 agaacatatc catcgcgtcc gccatctcca gcagccgcac gcggcgcatc tcgggcagcg
     4201 ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct
     4261 ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg
     4321 actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg
     4381 tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca
     4441 tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc
     4501 attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct
     4561 cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct
     4621 ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag
     4681 tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa
     4741 cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc
     4801 ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg
     4861 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4921 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     4981 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
     5041 gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     5101 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5161 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5221 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5281 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5341 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5401 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5461 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5521 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5581 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5641 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5701 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5761 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5821 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5881 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5941 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6001 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6061 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6121 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6181 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6241 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6301 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6361 gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6421 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6481 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6541 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6601 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6661 tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6721 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6781 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6841 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6901 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6961 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7021 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     7081 acctataaaa ataggcgtat cacgaggccc tttcgtctt
//
