LOCUS       sequence-273188-        5510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To facilitate restriction cloning of an RNA of interest with a TGT 
            recognition element in the 3' region.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5510)
  AUTHORS   Busby KN, Devaraj NK
  TITLE     Enzymatic covalent labeling of RNA with RNA transglycosylation at 
            guanosine (RNA-TAG).
  JOURNAL   Methods Enzymol. 2020;641:373-399. doi: 10.1016/bs.mie.2020.03.009. 
            Epub 2020 Apr 20.
  PUBMED    32713531
REFERENCE   2  (bases 1 to 5510)
  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: "Methods 
            Enzymol."; date: "2020"; volume: "641:373-399. doi"; pages: " 
            10.1016/bs.mie.2020.03.009. Epub 2020 Apr 20"
COMMENT     Sequence Label: pcDNA3.1-(empty)-TAG
FEATURES             Location/Qualifiers
     source          1..5510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1046..1063)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1046..1062)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1101..1118)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1107..1331
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1377..1805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1464..1483)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1674..1695
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1814..1834)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1819..2148
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1999..2134
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2061..2080
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2215..3009
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2269..2288)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2879..2898
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3183..3304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3220..3239)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3274..3293
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3353..3369)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3353..3369)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3366..3388)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3377..3393
                     /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        complement(join(3401..3407,3408..3425,3426..3431))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3446..3467
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3584..3601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3755..4343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3835..3854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4514..5305,5306..5374))
                     /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     5137..5156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5375..5479)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atcctctaac acagactctc ggtaccatca
      961 ttttcatatc cccgggagca gactgtaaat ctgctcccac caccatcatt taatgaattc
     1021 catcaggaat ccctcacttc tgcagactgg ccgtcgtttt acactcgagt ctagagggcc
     1081 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     1141 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1201 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     1261 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     1321 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     1381 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1441 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1501 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1561 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1621 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1681 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1741 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1801 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     1861 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     1921 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     1981 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2041 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     2101 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     2161 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     2221 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     2281 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     2341 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     2401 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     2461 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     2521 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     2581 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     2641 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     2701 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     2761 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     2821 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     2881 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     2941 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3001 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     3061 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3121 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3181 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3241 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3301 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3361 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3421 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3481 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3541 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3601 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3661 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3721 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3781 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3841 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3901 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3961 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4021 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4081 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4141 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4201 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4261 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4321 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4381 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4441 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4501 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4561 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4621 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4681 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4741 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4801 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4861 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4921 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4981 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5041 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5101 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5161 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5221 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5281 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5341 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5401 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5461 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
