LOCUS       sequence-347865-        6518 bp    DNA     circular SYN 15-DEC-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 6518)
  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 6518)
  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 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..6518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(37..696)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     611..630
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(750..756,757..774,775..780))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_RNA        857..875
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        896..914
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        966..984
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1005..1023
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1075..1093
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1114..1132
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1184..1202
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1223..1241
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1293..1311
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1332..1350
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1402..1420
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1441..1459
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(1548..1565)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1554..1778
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1788..1807)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1789..1807)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1817..1839)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2063..2086)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2413..2432
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2495..3094
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2495..2514)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2991..3011
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3137..3156)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3191..3210
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3254..3273)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3373..3395
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3433..3451)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3519..3623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3624..3692,3693..4484)
                     /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(3842..3861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4655..5243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5144..5163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5397..5414
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5531..5552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5567..5572,5573..5590,5591..5597)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5605..5621
                     /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        5635..5653
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5635..5652
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        5683..5894
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             complement(5908..6213)
                     /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(6010..6029)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
ORIGIN
        1 ctgttatctg gcttttagta agccggatcc acgcgtttac gccccgccct gccactcatc
       61 gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg
      121 atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat
      181 ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa
      241 actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata
      301 ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa
      361 atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt
      421 gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa
      481 ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg
      541 cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata
      601 ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat
      661 atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa
      721 tctcgacgga tcctaactca aaatccacac attatacgag ccggaagcat aaagtgtaaa
      781 gcctggggtg cctaatgcgg ccgcggtctc acccgggccc tatatatgga tcctaaggta
      841 cctaattgcc tagaaaacat gaggatcacc catgtctgca ggtcgactct agaaaacatg
      901 aggatcaccc atgtctgcag tattcccggg ttcattagat cctaaggtac ctaattgcct
      961 agaaaacatg aggatcaccc atgtctgcag gtcgactcta gaaaacatga ggatcaccca
     1021 tgtctgcagt attcccgggt tcattagatc ctaaggtacc taattgccta gaaaacatga
     1081 ggatcaccca tgtctgcagg tcgactccag aaaacatgag gatcacccat gtctgcagta
     1141 ttcccgggtt cattagatcc taaggtacct aattgcctag aaaacatgag gatcacccat
     1201 gtctgcaggt cgactctaga aaacatgagg atcacccatg tctgcagtat tcccgggttc
     1261 attagatcct aaggtaccta attgcctaga aaacatgagg atcacccatg tctgcaggtc
     1321 gactctagaa aacatgagga tcacccatgt ctgcagtatt cccgggttca ttagatccta
     1381 aggtacctaa ttgcctagaa aacatgagga tcacccatgt ctgcaggtcg actccagaaa
     1441 acatgaggat cacccatgtc tgcagtattc ccgggttcat tagatctgcg cgcgatcgat
     1501 atcagcggcc gcataatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     1561 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1621 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1681 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     1741 caatagcagg catgctgggg atgcggtggg ctctatggga attctcgccc tatagtgagt
     1801 cgtattacct ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     1861 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     1921 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt
     1981 ttctccttac gcatctgtgc ggtatttcac accgcatatg ttctaccggg taggggaggc
     2041 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
     2101 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
     2161 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
     2221 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
     2281 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
     2341 atagcagctt tgctccttcg ctttctgggc tcaggggcgg ggcgggcgcc cgaaggtcct
     2401 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
     2461 cctcatctcc gggcctttcg acctaacagc caccatgacc gagtacaagc ccacggtgcg
     2521 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     2581 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     2641 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     2701 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     2761 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     2821 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     2881 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     2941 ggcggccgag cgcgccgggg tgcccgcctt cctggaaacc tccgcgcccc gcaacctccc
     3001 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     3061 cacctggtgc atgacccgca agcccggtgc ctgaggctcc acttgtttat tgcagcttat
     3121 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3181 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctcatat
     3241 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     3301 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3361 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3421 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3481 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3541 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3601 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3661 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3721 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3781 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3841 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3901 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3961 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4021 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4081 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4141 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4201 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4261 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4321 ctggagccgg tgagcgtggc tctcgaggta tcattgcagc actggggcca gatggtaagc
     4381 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4441 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4501 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4561 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4801 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5281 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5341 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5401 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5461 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5521 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5581 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacacg agctatttag
     5641 gtgacactat agaagtgaag cttggctccg gtgcccgtca gtgggcagag cgcacatcgc
     5701 ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt
     5761 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     5821 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     5881 ccgccagaac acagggagac cgtcgactta tattccccag aacatcaggt taatggcgtt
     5941 tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa cggaaaccgg
     6001 cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat gcaccaccgg
     6061 gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg ggatcaccat
     6121 ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac gataacggct
     6181 ctctctttta taggtgtaaa ccttaaactg catttcacca gcccctgttc tcgtcagcaa
     6241 aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga ttttccgctt
     6301 tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt accagaccgg
     6361 agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt
     6421 aatacgctgc ttcatagcat acctcttttt gacatacttc gggtatacat atcagtatat
     6481 attcttatac cgcaaaaatc agcgcgcaaa tacgcata
//
