LOCUS       sequence-423978-        6743 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter system, NMD negative control. Intron present in
            5'UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6743)
  AUTHORS   Kolakada D, Campbell AE, Galvis LB, Li Z, Lore M, Jagannathan S
  TITLE     A system of reporters for comparative investigation of 
            EJC-independent and EJC-enhanced nonsense-mediated mRNA decay.
  JOURNAL   Nucleic Acids Res. 2024 Feb 20:gkae121. doi: 10.1093/nar/gkae121.
  PUBMED    38375914
REFERENCE   2  (bases 1 to 6743)
  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: "Nucleic 
            Acids Res. 2024 Feb 20:gkae121. doi: 10.1093/nar/gkae121."
COMMENT     Sequence Label: AiRTX_ctrl
FEATURES             Location/Qualifiers
     source          1..6743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    1049..1170
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1183..1216)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1430..1449
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1448..1503
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1549..1568
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(join(1645..2358,2359..2361,2362..2364))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNIYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFRTAAGITHGMDELYK"
     primer_bind     2039..2058
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2298..2319
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2361..2370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2397..2421)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     enhancer        2529..2832
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2833..3036
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2987..3007
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     ncRNA           3163..3189
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           3215..3256
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     primer_bind     3331..3350
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      3364..3373
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3373..3402
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3403..4101
                     /codon_start=1
                     /product="far-red-shifted derivative of red fluorescent 
                     protein from Entacmaea quadricolor, also known as Katushka 
                     (Shcherbo et al., 2007)"
                     /label=TurboFP635
                     /note="mammalian codon-optimized"
                     /translation="GEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTMKI
                     KVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVL
                     TATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHSQMA
                     LKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAVAR
                     YCDLPSKLGHS"
     polyA_signal    4168..4289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4385..4496
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4485..4502
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4638..5237)
                     /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(4721..4741)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5218..5237
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    complement(5242..5275)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     polyA_signal    5487..5535
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        5608..5712
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5713..5781,5782..6573)
                     /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(5931..5950)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tgggtaccta agatacattg atgagtttgg acaaaccaca
     1081 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     1141 gtaaccatta taagctgcaa taaacaagtt gacggtatcg taataacttc gtatagcata
     1201 cattatacga agttatatgg atccatatgc tagctcgacg ctcaggtcga gggatcttca
     1261 taagagaaga gggacagcta tgactgggag taaggaatgg acagcagggg gctgtttcat
     1321 atactgatga cctctttata gccacctttg ttcatggcag ccagcatatg gcatatgttg
     1381 ccaaactcta aaccaaatac tcattctgat gttttaaatg atttgccctc ccatatgtcc
     1441 ttccgagtga gagacacaaa aaattccaac acactattgc aatgaaaata aatttccttt
     1501 attagccaga agtcagatgc tcaaggggct tcatgatgtc cccataattt ttggcagagg
     1561 gaaaaagatc tcagtggtat ttgtgagcca gggcattggc cacaccagcc accaccttct
     1621 gataggcagc ctgcacctga gtgtttactt gtacagctcg tccatgccgt gagtgatccc
     1681 ggcggcggtg cggaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct
     1741 cagcttggac tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat
     1801 gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg
     1861 gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agatgttgtg
     1921 gctgttgaag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat
     1981 gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt
     2041 cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat
     2101 ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac
     2161 gccgtggctc agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca
     2221 gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct
     2281 gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa
     2341 cagctcctcg cccttgctca ccatggtggc gaccacgatc ctcatcctgt ctctgcgatc
     2401 tgacggttca ctaaacgagc tctgcttata taggcctccc accgtacacg ccacctcgac
     2461 atactcagta gttattaata gtaatcaatt acggggtcat tagttcatag cgaggctgca
     2521 gtctaccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2581 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2641 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2701 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2761 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2821 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     2881 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     2941 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     3001 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagataccg
     3061 gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc
     3121 gagacagaga agactcttgc gtttcggtcg acttctagag ctctggaggc ttgctgaagg
     3181 ctgtatgcta gagacgtaca gatgcgtctc acaggacaca aggcctgtta ctagcactca
     3241 catggaacaa atggccgaat tcggcgctag ctgctgatat cgcatacgcg tggaccagat
     3301 aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt gccggtcgcc
     3361 accgccacca tggaacaaaa gctgatttct gaagaagact tgggtgagga tagcgtgctg
     3421 atcaccgaga acatgcacat gaaactgtac atggagggca ccgtgaacga ccaccacttc
     3481 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gaagatcaag
     3541 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac
     3601 ggcagcaaaa cctttatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc
     3661 cctgagggct tcacatggga gaggatcacc acatacgaag acgggggcgt gctgaccgct
     3721 acccaggaca ccagcctcca gaacggctgc ctcatctaca acgtcaagat caacggggtg
     3781 aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccagcacc
     3841 gagatgctgt accccgctga cagcggcctg agaggccata gccagatggc cctgaagctc
     3901 gtgggcgggg gctacctgca ctgctccctc aagaccacat acagatccaa gaaacccgct
     3961 aagaacctca agatgcccgg cttctacttc gtggacagga gactggaaag aatcaaggag
     4021 gccgacaaag agacctacgt cgagcagcac gagatggctg tggctcgcta ctgcgacctg
     4081 cctagcaaac tggggcacag cctcgagggt ggttctggtg gttctgccta aggtggttct
     4141 ggtggttctg aattaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     4201 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4261 ggtttgtcca aactcatcaa tgtatcttaa cgcgaaacta gtgctattgt cttcccaatc
     4321 ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca
     4381 atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt
     4441 caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga
     4501 ggctgatcag cgagctctag agtcggtggg cctcgggggc gggtgcgggg tcggcggggc
     4561 cgccccgggt ggcttcggtc ggagccatgg ggtcgtgcgc tcctttcggt cgggcgctgc
     4621 gggtcgtggg gcgggcgtca ggcaccgggc ttgcgggtca tgcaccaggt gcgcggtcct
     4681 tcgggcacct cgacgtcggc ggtgacggtg aagccgagcc gctcgtagaa ggggaggttg
     4741 cggggcgcgg aggtctccag gaaggcgggc accccggcgc gctcggccgc ctccactccg
     4801 gggagcacga cggcgctgcc cagacccttg ccctggtggt cgggcgacac gccgacggtg
     4861 gccaggaacc acgcgggctc cttgggccgg tgcggcgcca ggaggccttc catctgttgc
     4921 tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg ggccgatctc ggcgaacacc
     4981 gcccccgctt cgacgctctc cggcgtggtc cagaccgcca ccgcggcgcc gtcgtccgcg
     5041 acccacacct tgccgatgtc gagcccgacg cgcgtgagga agagttcttg cagctcggtg
     5101 acccgctcga tgtggcggtc cgggtcgacg gtgtggcgcg tggcggggta gtcggcgaac
     5161 gcggcggcga gggtgcgtac ggcccggggg acgtcgtcgc gggtggcgag gcgcaccgtg
     5221 ggcttgtact cggtcatggt gataacttcg tataggatac tttatacgaa gttatttatt
     5281 ctcgaatggg ggaccccgtc cctaacccac ggggccagtg gctatggcag ggcctgccgc
     5341 cccgacgttg gctgcgagcc ctgggccttc acccgaactt ggggggtggg gtggggaaaa
     5401 ggaagaaacg cgggcgtatt ggccccaatg gggtctcggt ggggtatcga cagagtgcca
     5461 gccctgggac cgaaccccgc gtttatgaac aaacgaccca acacccgtgc gttttattct
     5521 gtctttttat tgccgtcata gcgcgggttc cttccggtat tgtctccttc cgtgtttatt
     5581 aagcttggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5641 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5701 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5761 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5821 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5881 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5941 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6001 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6061 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6121 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6181 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6241 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6301 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6361 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6421 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6481 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6541 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6601 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6661 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6721 gtagaaaaga tcaaaggatc ttc
//
