LOCUS       sequence-444181-        5219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid backbone with multiple cloning site for two genes in 
            opposing directions (convergently transcribed).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5219)
  AUTHORS   Ljiljana Mihajlovic, Bharat Ravi Iyengar, Florian Baier, Icvara 
            Barbier, Justyna Iwaszkiewicz, Vincent Zoete, Andreas Wagner, 
            Yolanda Schaerli
  TITLE     A direct experimental test of Ohno's hypothesis
  JOURNAL   eLife13:RP97216
REFERENCE   2  (bases 1 to 5219)
  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: 
            "eLife13:RP97216"
COMMENT     Sequence Label: pAND-MCS
FEATURES             Location/Qualifiers
     source          1..5219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(284..829)
                     /direction=LEFT
                     /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     complement(364..383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(918..1733)
                     /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(918..1733)
                     /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     1641..1660
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1725..1744
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1870..1887
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1871..1887
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1907..1925
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1932..1950
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1965..1987
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      2062..2089
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2183..2201)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             2212..2234
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2239..2258
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     promoter        complement(join(2264..2270,2271..2286,2287..2292))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             2413..3495
                     /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(2432..2451)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4306..4929
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4379..4398)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5112..5131)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cacgaggcag acctcagcgc tagcggagtg tatactggct tactatgttg gcactgatga
       61 gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag
      121 aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt
      181 cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg
      241 aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc
      301 cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac
      361 tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct
      421 ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca
      481 ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag
      541 tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca
      601 aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg
      661 cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt
      721 acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt
      781 tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct
      841 tattaagggg tctgacgctc agtggaacga aaaatcaatc taaagtatat atgagtaaac
      901 ttggtctgac agttacctta gaaaaactca tcgagcatca aatgaaactg caatttattc
      961 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     1021 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     1081 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     1141 tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag
     1201 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     1261 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     1321 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     1381 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     1441 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     1501 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     1561 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     1621 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     1681 ttggaattta atcgcggcct ggagcaagac gtttcccgtt gaatatggct cataacaccc
     1741 cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct
     1801 tgtgcaatgt acatcagaga ttttgagaca caaggtctcc cgctcgacgt acggtggaag
     1861 aattcgagtt gtaaaacgac ggccagtact ccaccgttgg cttttttccc tatcagtgat
     1921 agagattgac atccctatca gtgatagaga taatgagcac taattttgtt taactttaag
     1981 aaggagatat acatatggtt gagttacctg cagcgattgg agctccaatc gcttggacca
     2041 gctttccctg caggtcacac tggctcacct tcgggtgggc ctttctgcgt ttatatacta
     2101 gagagagaat ataaaaagcc agattattaa tccggctttt ttattatttg tcgacctcat
     2161 tcgctaatcg ccacggtacc caccgctgag caataactag cccatggtat atctccttct
     2221 taaagttaaa caaaattaaa ttgtgagcgc tcacaattcc acacattata cgagccgatg
     2281 attaattgtc aacagctcgt aaaagacgca gtgacggcgc ggccgcaatg gcggcgcgcc
     2341 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     2401 agggtggtga atatgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     2461 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     2521 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     2581 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     2641 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     2701 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     2761 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     2821 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     2881 aacagtatta ttttctccca tgaggacggt acgcgactgg gcgtggagca tctggtcgca
     2941 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     3001 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     3061 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     3121 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     3181 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     3241 gaagatagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     3301 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     3361 cagctgttgc cagtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     3421 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     3481 gaaagcgggc agtgataaga tcctattcca gcgggtcaca caggaaaggc ctcgatgaaa
     3541 aacataaatg ccgacgacac atacagaata attaataaaa ttaaagcttg tagaagcaat
     3601 aatgatatta atcaatgctt atctgatatg actaaaatgg tacattgtga atattattta
     3661 ctcgcgatca tttatcctca ttctatggtt aaatctgata tttcaatcct agataattac
     3721 cctaaaaaat ggaggcaata ttatgatgac gctaatttaa taaaatatga tcctatagta
     3781 gattattcta actccaatca ttcaccaatt aattggaata tatttgaaaa caatgctgta
     3841 aataaaaaat ctccaaatgt aattaaagaa gcgaaaacat caggtcttat cactgggttt
     3901 agtttcccta ttcatacggc taacaatggc ttcggaatgc ttagttttgc acattcagaa
     3961 aaagacaact atatagatag tttattttta catgcgtgta tgaacatacc attaattgtt
     4021 ccttctctag ttgataatta tcgaaaaata aatatagcaa ataataaatc aaacaacgat
     4081 ttaaccaaaa gagaaaaaga atgtttagcg tgggcatgcg aaggaaaaag ctcttgggat
     4141 atttcaaaaa tattaggttg cagtgagcgt actgtcactt tccatttaac caatgcgcaa
     4201 atgaaactca atacaacaaa ccgctgccaa agtatttcta aagcaatttt aacaggagca
     4261 attgattgcc catactttaa aaattaagga tccaggagga aaaaaatgtc cagattagat
     4321 aaaagtaaag tgattaacag cgcattagag ctgcttaatg aggtcggaat cgaaggttta
     4381 acaacccgta aactcgccca gaagctaggt gtagagcagc ctacattgta ttggcatgta
     4441 aaaaataagc gggctttgct cgacgcctta gccattgaga tgttagatag gcaccatact
     4501 cacttttgcc ctttagaagg ggaaagctgg caagattttt tacgtaataa cgctaaaagt
     4561 tttagatgtg ctttactaag tcatcgcgat ggagcaaaag tacatttagg tacacggcct
     4621 acagaaaaac agtatgaaac tctcgaaaat caattagcct ttttatgcca acaaggtttt
     4681 tcactagaga atgcattata tgcactcagc gctgtggggc attttacttt aggttgcgta
     4741 ttggaagatc aagagcatca agtcgctaaa gaagaaaggg aaacacctac tactgatagt
     4801 atgccgccat tattacgaca agctatcgaa ttatttgatc accaaggtgc agagccagcc
     4861 ttcttattcg gccttgaatt gattatatgc ggattagaaa aacaacttaa atgtgaaagt
     4921 gggtcctaat aattggtaac gaatcagaca attgacggct cgagggagta gcatagggtt
     4981 tgcagaatcc ctgcttcgtc catttgacag gcacattatg catcgatgat aagctgtcaa
     5041 acatgagcag atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     5101 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     5161 gctcatgagc aaatatttta tctgaggtag agaccgcttc ctcgctcact gactcgctg
//
