LOCUS       sequence-86408-e        4075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces one half (alpha) of aminoglycoside phosphotransferase 
            (3′)-IIa when in the presence of T7 RNA Polymerase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4075)
  AUTHORS   Schmidt CM, Shis DL, Nguyen-Huu TD, Bennett MR
  TITLE     Stable maintenance of multiple plasmids in E. coli using a single 
            selective marker.
  JOURNAL   ACS Synth Biol. 2012 Oct 19;1(10):445-50. doi: 10.1021/sb3000589. 
            Epub 2012 Sep 4.
  PUBMED    23656183
REFERENCE   2  (bases 1 to 4075)
  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: "10.1021/sb3000589";
            journalName: "ACS Synth Biol"; date: "2012-10-19- 19"; volume: "1"; 
            issue: "10"; pages: "445-50"
COMMENT     Sequence Label: pCS4
FEATURES             Location/Qualifiers
     source          1..4075
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             255..632
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     primer_bind     complement(289..308)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1095..1640
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     1541..1560
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1757..1774
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(1816..1863)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1859..1877
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1930..1947)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2021..2032)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     2205..2224
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     RBS             2285..2307
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2322..2346
                     /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(2346..2365)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2347..2365)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2449..2468)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2674..2751
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2752..3834
                     /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(2771..2790)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4041..4075,1..70)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
       61 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      121 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      181 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      241 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
      301 ggtattatcc cgtttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
      361 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
      421 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
      481 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
      541 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
      601 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
      661 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
      721 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
      781 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
      841 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
      901 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
      961 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     1021 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agacccctta
     1081 ataagatgat cttcttgaga tcgttttggt ctgcgcgtaa tctcttgctc tgaaaacgaa
     1141 aaaaccgcct tgcagggcgg tttttcgaag gttctctgag ctaccaactc tttgaaccga
     1201 ggtaactggc ttggaggagc gcagtcacca aaacttgtcc tttcagttta gccttaaccg
     1261 gcgcatgact tcaagactaa ctcctctaaa tcaattacca gtggctgctg ccagtggtgc
     1321 ttttgcatgt ctttccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1381 ggactgaacg gggggttcgt gcatacagtc cagcttggag cgaactgcct acccggaact
     1441 gagtgtcagg cgtggaatga gacaaacgcg gccataacag cggaatgaca ccggtaaacc
     1501 gaaaggcagg aacaggagag cgcacgaggg agccgccagg gggaaacgcc tggtatcttt
     1561 atagtcctgt cgggtttcgc caccactgat ttgagcgtca gatttcgtga tgcttgtcag
     1621 gggggcggag cctatggaaa aacggctttg ccgcggccct ctcacttccc tgttaagtat
     1681 cttcctggca tcttccagga aatctccgcc ccgttcgtaa gccatttccg ctcgccgcag
     1741 tcgaacgacc gagcgtagcg agtcagtgag cgaggaagcg gaatatatcc tgtcggatat
     1801 agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc
     1861 tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc
     1921 cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt cgacggagct
     1981 cgaattcgga tccttactga gccagttctt tcttcagtgc ctgcagttcc cacttcagct
     2041 gagccagctc tttcttgttt gcctgcagct ctttcttaag ctgagcgcca gagccaccag
     2101 gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg
     2161 acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga
     2221 atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaaccatgg
     2281 tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac
     2341 aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg
     2401 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     2461 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
     2521 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac
     2581 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
     2641 aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt
     2701 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     2761 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     2821 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     2881 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     2941 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     3001 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     3061 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     3121 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     3181 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     3241 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     3301 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     3361 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     3421 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     3481 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     3541 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     3601 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     3661 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     3721 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     3781 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     3841 cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag
     3901 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     3961 tatgactgtc ttctttatca tgcaactcgt aggacaggtg cgtttcttag acgtcaggtg
     4021 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa ataca
//
