LOCUS       sequence-451687-        5180 bp    DNA     circular SYN 15-APR-2025
DEFINITION  In vitro transcription of cap-dependently translated mRNA encoding 
            mCherry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5180)
  TITLE     Enhancing cap-independent translation of mRNA via in vitro 
            transcription priming
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5180)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pmRNA-mCherry-A120
FEATURES             Location/Qualifiers
     source          1..5180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      74..662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     563..582
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        873..1252
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1253..1456
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1407..1427
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1453..1477
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1592..1724
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1707..1726
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    1823..1903
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1905..1914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1911..2621
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2056..2074)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2324..2343
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3095..3216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3106..3125)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3160..3179
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3397..3852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3484..3503)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3694..3715
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3919..3938)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4038..4060
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4098..4116)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4184..4288
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4289..5083
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4289..5083
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4343..4362)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4953..4972
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
       61 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      121 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      181 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      241 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      301 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      361 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      421 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      481 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      541 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      601 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      661 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      721 tggctcgaca gatcatcaat attggccatt agccatatta ttcattggtt atatagcata
      781 aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta
      841 tattggctca tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata
      901 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      961 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     1021 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     1081 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc
     1141 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg
     1201 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     1261 gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     1321 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     1381 atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt
     1441 ctatataagc agagctcgtt tagtgaaccg tcagatcact agacgcttta ttgcggtagt
     1501 ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct cgaacttaag
     1561 ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta
     1621 aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc
     1681 acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt
     1741 caattacagc tcttaaggct agagtcacct gcaacatgac ttaatacgac tcactataag
     1801 gagagatatc acgcgttcta gagctagcgc taccggactc agatctcgag ctcaagcttc
     1861 gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcgccacc atggtgagca
     1921 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     1981 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     2041 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     2101 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     2161 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     2221 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     2281 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     2341 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     2401 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     2461 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     2521 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     2581 gccgccactc caccggcggc atggacgagc tgtacaagta aagcggccgc agctcgcttt
     2641 cttgctgtcc aatttctatt aaaggttcct ttgttcccta agtccaacta ctaaactggg
     2701 ggatattatg aagggccttg agcatttgga ttctgcctaa taaaaaacat ttattttcat
     2761 tgcgtttagc tcgctttctt gctgtccaat ttctattaaa ggttcctttg ttccctaagt
     2821 ccaactacta aactggggga tattatgaag ggccttgagc atttggattc tgcctaataa
     2881 aaaacattta ttttcattgc atttaaatgt ttaaacaaaa aaaaaaaaaa aaaaaaaaaa
     2941 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3001 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcaggtggga gggtacgttc
     3061 gtgataaaca aacggccgct tcgagcagac atgataagat acattgatga gtttggacaa
     3121 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     3181 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     3241 atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa
     3301 tgtggtaaaa tcgataagga tcagggctgg cgtaatagcg aagaggcccg caccgatcgc
     3361 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
     3421 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3481 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3541 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3601 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3661 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3721 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3781 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3841 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3901 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3961 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4021 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4081 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4141 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4201 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4261 gcttcaataa tattgaaaaa ggaagagtat gattgaacaa gatggattgc acgcaggttc
     4321 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4381 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4441 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     4501 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4561 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4621 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4681 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     4741 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     4801 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     4861 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     4921 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4981 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     5041 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgactgtcag accaagttta
     5101 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5161 gatccttttt gataatctca
//
