LOCUS       sequence-306062-        6947 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MSCV backbone with murine TCR-alpha signal peptide and murine 
            TCR-beta2 constant region (TRBC2) for expression of TCR genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6947)
  AUTHORS   Anderson AM, Landry LG, Alkanani AA, Pyle L, Powers AC, Atkinson MA,
            Mathews CE, Roep BO, Michels AW, Nakayama M
  TITLE     Human islet T cells are highly reactive to preproinsulin in type 1 
            diabetes.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Oct 12;118(41). pii: 2107208118. doi:
            10.1073/pnas.2107208118.
  PUBMED    34611019
REFERENCE   2  (bases 1 to 6947)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2021-10-12"; volume: "118(41). pii: 
            2107208118. doi"; pages: " 10.1073/pnas.2107208118"
COMMENT     Sequence Label: MBC2-PuroIII
FEATURES             Location/Qualifiers
     source          1..6947
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             230..746
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    810..1151
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1218..1634
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1564..1586
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1602..1618
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    2276..2825
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2420..2437)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2647..2666
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2828..3427
                     /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(2828..2847)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3324..3344
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3571..4085
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4247..4263
                     /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(4271..4277,4278..4295,4296..4301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4316..4337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4454..4471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4625..5213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4705..4724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5384..6175,6176..6244))
                     /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     6007..6026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6245..6349)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6417..6435
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6473..6495)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6595..6614
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6808..6830
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
       61 tgccggccac gatgcgtccg gcgtagaggc gattagtcca atttgttaaa gacaggatat
      121 cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg
      181 agccatagat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
      241 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaatac
      301 ataactgaga atagagaagt tcagatcaag gttaggaaca gagagacagc agaatatggg
      361 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt
      421 ccccagatgc ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc
      481 cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc
      541 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg
      601 cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat aaagcctctt
      661 gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc agattgattg
      721 actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag acccctgccc
      781 agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc gtgtctgtct
      841 ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg tactagttag
      901 ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga acacccggcc
      961 gcaaccctgg gagacgtccc agggactttg ggggccgttt ttgtggcccg acctgaggaa
     1021 gggagtcgat gtggaatccg accccgtcag gatatgtggt tctggtagga gacgagaacc
     1081 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttggaaccga agccgcgcgt
     1141 cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact gtgtttctgt
     1201 atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc ttaggtcact
     1261 ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tgtcaagaag agacgttggg
     1321 ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg atggccgcga gacggcacct
     1381 ttaaccgaga cctcatcacc caggttaaga tcaaggtctt ttcacctggc ccgcatggac
     1441 acccagacca ggtcccctac atcgtgacct gggaagcctt ggcttttgac ccccctccct
     1501 gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc gccccgtctc
     1561 tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cctttatcca gccctcactc
     1621 cttctctagg cgccggaatt cacgcgtcac catgaaaaca tatgctccta cattattcat
     1681 gtttctatgg ctgcagctgg atgggatgag tcgacgtacc agttagatct gagaaatgtg
     1741 actccaccca aggtctcctt gtttgagcca tcaaaagcag agattgcaaa caaacaaaag
     1801 gctaccctcg tgtgcttggc caggggcttc ttccctgacc acgtggagct gagctggtgg
     1861 gtgaatggca aggaggtcca cagtggggtc agcacggacc ctcaggccta caaggagagc
     1921 aattatagct actgcctgag cagccgcctg agggtctctg ctaccttctg gcacaatcct
     1981 cgaaaccact tccgctgcca agtgcagttc catgggcttt cagaggagga caagtggcca
     2041 gagggctcac ccaaacctgt cacacagaac atcagtgcag aggcctgggg ccgagcagac
     2101 tgtggaatca cttcagcatc ctatcatcag ggggttctgt ctgcaaccat cctctatgag
     2161 atcctactgg ggaaggccac cctatatgct gtgctggtca gtggcctggt gctgatggcc
     2221 atggtcaaga aaaaaaattc ctgactcgag cgggatcaat tccgcccccc ccctaacgtt
     2281 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     2341 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     2401 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     2461 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     2521 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     2581 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     2641 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     2701 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     2761 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     2821 taataccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     2881 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     2941 tccagaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     3001 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     3061 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     3121 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     3181 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     3241 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     3301 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     3361 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     3421 tgcctgactc gacctgcagg catgcaagct tcaggtagcc ggctaacgtt aacaaccggt
     3481 acctctagaa ctatagctag catgcgcaaa tttaaagcgc tgatatcgat aaaataaaag
     3541 attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag
     3601 ctagcttaag taacgccatt ttgcaaggca tggaaaatac ataactgaga atagagaagt
     3661 tcagatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga tatctgtggt
     3721 aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc
     3781 tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc
     3841 ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc
     3901 tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc ctccgataga
     3961 ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg
     4021 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct
     4081 ttcatgggta acagtttctt gaagttggag aacaacattc tgagggtagg agtcgaatat
     4141 taagtaatcc tgactcaatt agccactgtt ttgaatccac atactccaat actcctgaaa
     4201 tagttcatta tggacagcgc agaaagagct ggggagaatt gtgaaattgt tatccgctca
     4261 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4321 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4381 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4441 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4501 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4561 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4621 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4681 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4741 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4801 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4861 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4921 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4981 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5041 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5101 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5161 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5221 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5281 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5341 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5401 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5461 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5521 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5581 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5641 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5701 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5761 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5821 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5881 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5941 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6001 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6061 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6121 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6181 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6241 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6301 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6361 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     6421 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     6481 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     6541 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     6601 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     6661 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     6721 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     6781 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggcgcaag
     6841 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     6901 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgat
//
