LOCUS       sequence-430720-        6363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses human MR1 resistant to CRISPR editing with a specific 
            sgRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6363)
  AUTHORS   Kulicke CA, Swarbrick GM, Ladd NA, Cansler M, Null M, Worley A, 
            Lemon C, Ahmed T, Bennett J, Lust TN, Heisler CM, Huber ME, Krawic 
            JR, Ankley LM, McBride SK, Tafesse FG, Olive AJ, Hildebrand WH, 
            Lewinsohn DA, Adams EJ, Lewinsohn DM, Harriff MJ
  TITLE     Delivery of loaded MR1 monomer results in efficient ligand exchange 
            to host MR1 and subsequent MR1T cell activation.
  JOURNAL   Commun Biol. 2024 Feb 24;7(1):228. doi: 10.1038/s42003-024-05912-4.
  PUBMED    38402309
REFERENCE   2  (bases 1 to 6363)
  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: "Commun 
            Biol."; date: "2024-02-24"; volume: "7(1):228. doi"; pages: " 
            10.1038/s42003-024-05912-4"
COMMENT     Sequence Label: pCI AscI MR1 res
FEATURES             Location/Qualifiers
     source          1..6363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..62)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     253..274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(478..497)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     597..619
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(657..675)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        743..847
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(848..916,917..1708)
                     /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(1066..1085)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1879..2467
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2368..2387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2689..3068
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3069..3272
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3223..3243
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3269..3293
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          3408..3540
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3523..3542
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        3585..3603
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4682..5256,5257..5259,5260..5268)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4849..4866)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5076..5095
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(5269..5271,5272..5274,5275..5988)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5269..5271,5272..5274,5275..5985)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5314..5335)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5575..5594)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5922..5943
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6017..6138
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6028..6047)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6082..6101
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      join(6319..6363,1..411)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
       61 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      121 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      181 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      241 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
      301 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
      361 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg
      421 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
      481 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
      541 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
      601 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
      661 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
      721 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
      781 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
      841 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
      901 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
      961 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     1021 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     1081 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     1141 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     1201 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     1261 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     1321 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     1381 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     1441 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     1501 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     1561 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     1621 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     1681 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     1741 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     1801 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1861 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1921 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1981 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     2041 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2101 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2161 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2221 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2281 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2341 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2401 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2461 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     2521 tcacatggct cgacaggcgc gccggccggc ctcaatattg gccattagcc atattattca
     2581 ttggttatat agcataaatc aatattggct attggccatt gcatacgttg tatctatatc
     2641 ataatatgta catttatatt ggctcatgtc caatatgacc gccatgttgg cattgattat
     2701 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     2761 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     2821 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     2881 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     2941 tgccaagtcc gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     3001 agtacatgac cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     3061 ttaccatggt gatgcggttt tggcagtaca ccaatgggcg tggatagcgg tttgactcac
     3121 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     3181 aacgggactt tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc
     3241 gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcactagaa
     3301 gctttattca ccggtgttat cacagttaaa ttgctaacgc agtcagtgct tctgacacaa
     3361 cagtctcgaa cttaagctgc agaagttggt cgtgaggcac tgggcaggta agtatcaagg
     3421 ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag agaagactct
     3481 tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacag
     3541 gtgtccactc ccagttcaat tacagctctt aaggctagag tacttaatac gactcactat
     3601 aggctagcct cgagaattca tgggggaact gatggcgttc ctgttacctc tcatcattgt
     3661 gttaatggtg aagcacagcg attcccggac gcactctctg agatattttc gcctgggcgt
     3721 cagcgatccc atccatgggg tccctgaatt tatttcggtt gggtacgtgg actcgcaccc
     3781 tatcaccaca tatgacagtg tcactcggca gaaggagcca cgggccccat ggatggcaga
     3841 gaacctcgcg cctgatcact gggagaggta cactcagctg ctgaggggct ggcagcagat
     3901 gttcaaggtg gaactgaagc gcctacagag gcactacaat cactcagggt ctcacactta
     3961 ccagagaatg attggctgtg agctgctgga ggatggaagc accacaggat ttctgcagta
     4021 tgcatatgac gggcaggatt tcctgatctt caataaagac accctctcct ggctggctgt
     4081 agataatgtg gctcacacca tcaagcaggc atgggaggcc aatcagcatg agttgctgta
     4141 tcaaaagaat tggctggaag aagaatgtat tgcctggcta aagagattcc tggagtatgg
     4201 gaaagacacc ctacaaagaa cagagccccc actggtcaga gtaaatcgca aagaaacttt
     4261 tccaggggtt acagctctct tctgcaaagc tcatggcttt taccccccag aaatttacat
     4321 gacatggatg aaaaacgggg aagaaattgt ccaagaaatt gattatggag acattcttcc
     4381 cagtggggat ggaacctatc aggcgtgggc atcaattgag cttgatcctc agagcagcaa
     4441 cctttactcc tgtcatgtgg agcactgcgg tgtccacatg gttcttcagg tcccccagga
     4501 atcagaaact atccctcttg tgatgaaagc tgtctctggg tccattgtcc ttgtcattgt
     4561 gctggctgga gttggtgttc tagtctggag aagaaggccc cgagagcaaa atggagccat
     4621 ctaccttcca acaccagatc gatgacggcc aattctgcag tcgacggtac cgcgggatcc
     4681 gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt
     4741 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc
     4801 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag
     4861 gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac
     4921 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc
     4981 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc
     5041 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
     5101 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt
     5161 gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg
     5221 gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat ggtgagcaag
     5281 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     5341 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     5401 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     5461 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     5521 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     5581 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     5641 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     5701 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     5761 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     5821 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     5881 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     5941 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaacc cgggcggccg
     6001 cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     6061 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     6121 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     6181 gagatgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag
     6241 gatccgggct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     6301 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6361 tta
//
