LOCUS       sequence-345021-        6497 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Assembly backbone for tagging RNA transcript with a 12X tandem MS2 
            stemloop at the 3'.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6497)
  AUTHORS   Delhaye L, De Bruycker E, Volders PJ, Fijalkowska D, De Sutter D, 
            Degroeve S, Martens L, Mestdagh P, Eyckerman S
  TITLE     Orthogonal proteomics methods to unravel the HOTAIR interactome.
  JOURNAL   Sci Rep. 2022 Jan 27;12(1):1513. doi: 10.1038/s41598-022-05405-6.
  PUBMED    35087108
REFERENCE   2  (bases 1 to 6497)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2022-01-27"; volume: "12(1):1513. doi"; pages: " 
            10.1038/s41598-022-05405-6"
COMMENT     Sequence Label: pEFS-ccdB-12X MS2_mPGK-Puro
FEATURES             Location/Qualifiers
     source          1..6497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(17..36)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     136..158
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(196..214)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        282..386
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(387..455,456..1247)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(605..624)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1418..2006
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1907..1926
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2160..2177
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2294..2315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2330..2335,2336..2353,2354..2360)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2368..2384
                     /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)."
     promoter        2398..2416
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2398..2415
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        2446..2657
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             complement(2671..2976)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2773..2792)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3318..3977)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3892..3911
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(4031..4037,4038..4055,4056..4061))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_RNA        4138..4156
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4177..4195
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4247..4265
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4286..4304
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4356..4374
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4395..4413
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4465..4483
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4504..4522
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4574..4592
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4613..4631
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4683..4701
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4722..4740
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(4808..4825)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4814..5038
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5048..5067)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5049..5067)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5077..5099)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5323..5346)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5673..5692
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5755..6354
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLDTS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5755..5774)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6251..6271
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6397..6416)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6451..6470
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
       61 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
      121 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
      181 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
      241 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      301 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
      361 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
      421 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
      481 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
      541 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
      601 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
      661 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
      721 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
      781 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
      841 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
      901 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
      961 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1021 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1081 aatctggagc cggtgagcgt ggctctcgag gtatcattgc agcactgggg ccagatggta
     1141 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1201 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     1261 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1321 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     1381 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1441 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1501 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1561 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1621 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1681 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1741 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1801 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1861 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1921 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     1981 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2041 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2101 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2161 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     2221 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     2281 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     2341 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acgagctatt
     2401 taggtgacac tatagaagtg aagcttggct ccggtgcccg tcagtgggca gagcgcacat
     2461 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa
     2521 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     2581 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     2641 ttgccgccag aacacaggga gaccgtcgac ttatattccc cagaacatca ggttaatggc
     2701 gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac
     2761 tggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac
     2821 cgggtaaagt tcacgggaga ctttatctga cagcagacgt gcactggcca gggggatcac
     2881 catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg
     2941 gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagtccctg ttctcgtcag
     3001 caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc tgattttccg
     3061 ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg cttaccagac
     3121 cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt caactgtcac
     3181 tgtaatacgc tgcttcatag cacacctctt tttgacatac ttcgggtata catatcagta
     3241 tatattctta taccgcaaaa atcagcgcgc aaatacgcat actgttatct ggcttttagt
     3301 aagccggatc cacgcgttta cgccccgccc tgccactcat cgcagtactg ttgtaattca
     3361 ttaagcattc tgccgacatg gaagccatca cagacggcat gatgaacctg aatcgccagc
     3421 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     3481 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     3541 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     3601 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     3661 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     3721 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc
     3781 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     3841 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     3901 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     3961 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgccgg atcctaactc
     4021 aaaatccaca cattatacga gccggaagca taaagtgtaa agcctggggt gcctaatgcg
     4081 gccgcggtct cacccgggcc ctatatatgg atcctaaggt acctaattgc ctagaaaaca
     4141 tgaggatcac ccatgtctgc aggtcgactc tagaaaacat gaggatcacc catgtctgca
     4201 gtattcccgg gttcattaga tcctaaggta cctaattgcc tagaaaacat gaggatcacc
     4261 catgtctgca ggtcgactct agaaaacatg aggatcaccc atgtctgcag tattcccggg
     4321 ttcattagat cctaaggtac ctaattgcct agaaaacatg aggatcaccc atgtctgcag
     4381 gtcgactcca gaaaacatga ggatcaccca tgtctgcagt attcccgggt tcattagatc
     4441 ctaaggtacc taattgccta gaaaacatga ggatcaccca tgtctgcagg tcgactctag
     4501 aaaacatgag gatcacccat gtctgcagta ttcccgggtt cattagatcc taaggtacct
     4561 aattgcctag aaaacatgag gatcacccat gtctgcaggt cgactctaga aaacatgagg
     4621 atcacccatg tctgcagtat tcccgggttc attagatcct aaggtaccta attgcctaga
     4681 aaacatgagg atcacccatg tctgcaggtc gactccagaa aacatgagga tcacccatgt
     4741 ctgcagtatt cccgggttca ttagatggcc gcataatcta gagggcccgt ttaaacccgc
     4801 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4861 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4921 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4981 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggga
     5041 attctcgccc tatagtgagt cgtattacct ggccgtcgtt ttacaacgtc gtgactggga
     5101 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     5161 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5221 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     5281 ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc
     5341 ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc
     5401 ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc
     5461 ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa
     5521 gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta
     5581 ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcaggggcgg
     5641 ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc
     5701 tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctaacagc caccatgacc
     5761 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     5821 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     5881 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     5941 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     6001 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     6061 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     6121 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     6181 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggacacc
     6241 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     6301 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaggctcc
     6361 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     6421 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     6481 atcatgtctg gctctca
//
