LOCUS       sequence-86406-e        5262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces 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 5262)
  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 5262)
  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: pCS1
FEATURES             Location/Qualifiers
     source          1..5262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(7..54)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     50..68
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(121..138)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(256..1071)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MGHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(256..1071)
                     /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="MGHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     979..998
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     RBS             1078..1100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1115..1139
                     /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(1139..1158)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1140..1158)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1242..1261)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1467..1544
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1545..2627
                     /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(1564..1583)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2894..3085
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3079..3101)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3187..3327
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3242..3261
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3342..3359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3513..4101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3593..3612)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4272..5063,5064..5132))
                     /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     4895..4914
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5133..5237)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtccaaa aaacccctca agacccgttt agaggcccca aggggttatg ctagttattg
       61 ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag ccggatctca
      121 gtggtggtgg tggtggtgct cgagtgcggc cgcaagcttg tcgacggagc tcgaattcgg
      181 atccatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      241 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
      301 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
      361 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
      421 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
      481 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
      541 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
      601 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
      661 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
      721 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
      781 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
      841 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
      901 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
      961 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     1021 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggccca tggtatatct
     1081 ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc
     1141 ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat
     1201 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     1261 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     1321 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     1381 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     1441 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
     1501 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     1561 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     1621 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     1681 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     1741 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     1801 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     1861 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     1921 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     1981 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     2041 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     2101 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     2161 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     2221 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     2281 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     2341 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     2401 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     2461 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     2521 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     2581 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     2641 aatccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     2701 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     2761 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     2821 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     2881 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     2941 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     3001 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     3061 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3121 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3181 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3241 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     3301 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     3361 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3421 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3481 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3541 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3601 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3661 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3721 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3781 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3841 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3901 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     3961 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4021 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4081 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4141 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4201 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4261 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4321 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4381 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4441 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4501 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4561 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     4621 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4681 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4741 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4801 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4861 cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact
     4921 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4981 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5041 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5101 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5161 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5221 caaatagggg ttccgcgcac atttccccga aaagtgccac ct
//
