LOCUS       pTwist_(CMV)-mCh        5800 bp    DNA     circular SYN 05-MAR-2019
DEFINITION  mCherry-CD8-OLLAS-GCN4 in pTwist CMV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5800)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 11, 2025 from SnapGene 8.1.0
            https://www.snapgene.com
COMMENT     This file was created using tools provided by Twist Bioscience
FEATURES             Location/Qualifiers
     source          1..5800
                     /organism="mCherry-CD8-OLLAS-GCN4 in pTwist CMV"
     promoter        2..20
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             171..878
                     /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"
     CDS             1488..1529
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     CDS             1554..1610
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     polyA_signal    1669..1790
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     terminator      1904..1951
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1981..2117
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2162..2233
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2459..3047)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             join(3793..3861,3862..4653)
                     /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"
     terminator      4666..4802
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    4954..5025
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      5030..5077
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    5094..5142
                     /note="synthetic polyadenylation signal"
     enhancer        5223..5526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5527..5730
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc ccatcgattc
       61 gaattcaagg cctctcgagc aaaatggcct caccgttgac ccgctttctg tcgctgaacc
      121 tgctgctgct gggtgagtcg attatcctgg ggagtggaga agctggtacc atggtgagca
      181 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
      241 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
      301 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
      361 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
      421 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
      481 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
      541 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
      601 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
      661 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
      721 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
      781 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
      841 gccgccactc caccggcggc atggacgagc tgtacaaggc atgcaagcca caggcacccg
      901 aactccgaat ctttccaaag aaaatggacg ccgaacttgg tcagaaggtg gacctggtat
      961 gtgaagtgtt ggggtccgtt tcgcaaggat gctcttggct cttccagaac tccagctcca
     1021 aactccccca gcccaccttc gttgtctata tggcttcatc ccacaacaag ataacgtggg
     1081 acgagaagct gaattcgtcg aaactgtttt ctgccatgag ggacacgaat aataagtacg
     1141 ttctcaccct gaacaagttc agcaaggaaa acgaaggcta ctatttctgc tcagtcatca
     1201 gcaactcggt gatgtacttc agttctgtcg tgccagtcct tcagaaagtg aactctacta
     1261 ctaccaagcc agtgctgcga actccctcac ctgtgcaccc taccgggaca tctcagcccc
     1321 agagaccaga agattgtcgg ccccgtggct cagtgaaggg gaccggattg gacttcgcct
     1381 gtgatattta catctgggca cccttggccg gaatctgcgt ggcccttctg ctgtccttga
     1441 tcatcactct catctgctac cacagccgcg gatccaccgg tatggggtcc ggatttgcca
     1501 atgaacttgg acctagactt atgggtaaaa ccggtactag tggaggtggc agcgaggaat
     1561 tgctatcgaa aaattaccat ttagaaaatg aggttgctag gctcaaaaag actagttagg
     1621 ctagcttgac tgactgagat acagcgtacc ttcagctcac agacatgata agatacattg
     1681 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     1741 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     1801 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     1861 aaaacctcta caaatgtggt attggcccat ctctatcggt atcgtagcat aaccccttgg
     1921 ggcctctaaa cgggtcttga ggggtttttt gtgcccctcg ggccggattg ctatctaccg
     1981 gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc
     2041 cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac
     2101 cagaaattta tccttaaggt cgtcagctat cctgcaggcg atctctcgat ttcgatcaag
     2161 acattccttt aatggtcttt tctggacacc actaggggtc agaagtagtt catcaaactt
     2221 tcttccctcc ctaatctcat tggttacctt gggctatcga aacttaatta accagtcaag
     2281 tcagctactt ggcgagatcg acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc
     2341 aagttcgatc tggtccttgc tattgcaccc gttctccgat tacgagtttc atttaaatca
     2401 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2461 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2521 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2581 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2641 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2701 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2761 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2821 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2881 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2941 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3001 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3061 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3121 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3181 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3241 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcatttaaa tttccgaact
     3301 ctccaaggcc ctcgtcggaa aatcttcaaa cctttcgtcc gatccatctt gcaggctacc
     3361 tctcgaacga actatcgcaa gtctcttggc cggccttgcg ccttggctat tgcttggcag
     3421 cgcctatcgc caggtattac tccaatcccg aatatccgag atcgggatca cccgagagaa
     3481 gttcaaccta catcctcaat cccgatctat ccgagatccg aggaatatcg aaatcggggc
     3541 gcgcctggtg taccgagaac gatcctctca gtgcgagtct cgacgatcca tatcgttgct
     3601 tggcagtcag ccagtcggaa tccagcttgg gacccaggaa gtccaatcgt cagatattgt
     3661 actcaagcct ggtcacggca gcgtaccgat ctgtttaaac ctagatattg atagtctgat
     3721 cggtcaacgt ataatcgagt cctagctttt gcaaacatct atcaagagac aggatcagca
     3781 ggaggctttc gcatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3841 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3901 cagttgggtg cgcgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3961 agttttcgcc ccgaagaacg ctttccaatg atgagcactt ttaaagttct gctatgtggc
     4021 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4081 cagaatgact tggttgagta ttcaccagtc acagaaaagc atcttacgga tggcatgaca
     4141 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4201 ctgacaacga ttggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4261 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4321 gacaccacga tgcctgtagc aatggcaaca accttgcgta aactattaac tggcgaacta
     4381 cttactctag cttcccggca acagttgata gactggatgg aggcggataa agttgcagga
     4441 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4501 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4561 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4621 gagataggtg cctcactgat taagcattgg taaccgattc taggtgcatt ggcgcagaaa
     4681 aaaatgcctg atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg
     4741 atacggcttc cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt
     4801 aagatcccga atcgtttaaa ctcgactctg gctctatcga atctccgtcg tttcgagctt
     4861 acgcgaacag ccgtggcgct catttgctcg tcgggcatcg aatctcgtca gctatcgtca
     4921 gcttaccttt ttggcagcga tcgcggctcc cgacatcttg gaccattagc tccacaggta
     4981 tcttcttccc tctagtggtc ataacagcag cttcagctac ctctcaattc aaaaaacccc
     5041 tcaagacccg tttagaggcc ccaaggggtt atgctatcaa tcgttgcgtt acacacacaa
     5101 aaaaccaaca cacatccatc ttcgatggat agcgatttta ttatctaact gctgatcgag
     5161 tgtagccaga tctagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     5221 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     5281 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     5341 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     5401 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     5461 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     5521 accatgctga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     5581 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     5641 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     5701 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat cagatctttg
     5761 tcgatcctac catccactcg acacacccgc cagcggccgc
//
