LOCUS       sequence-306221-        6947 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MSCV backbone with murine TCR-alpha signal peptide and murine 
            TCR-beta1 constant region (TRBC1) 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: MBC1-PuroIII
FEATURES             Location/Qualifiers
     source          1..6947
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             86..602
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    666..1007
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1074..1490
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1420..1442
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1458..1474
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    2132..2681
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2276..2293)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2503..2522
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2684..3283
                     /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(2684..2703)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3180..3200
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3427..3941
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4103..4119
                     /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(4127..4133,4134..4151,4152..4157))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4172..4193
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4310..4327)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4481..5069)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4561..4580)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5240..6031,6032..6100))
                     /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     5863..5882
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6101..6205)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6273..6291
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6329..6351)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6451..6470
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6664..6686
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6814..6833
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa taaaagattt
       61 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag
      121 cttaagtaac gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag
      181 atcaaggtta ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc
      241 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag
      301 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt
      361 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc
      421 cgagctcaat aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc
      481 gtcgcccggg tacccgtatt cccaataaag cctcttgctg tttgcatccg aatcgtggac
      541 tcgctgatcc ttgggagggt ctcctcagat tgattgactg cccacctcgg gggtctttca
      601 tttggaggtt ccaccgagat ttggagaccc ctgcccaggg accaccgacc cccccgccgg
      661 gaggtaagct ggccagcggt cgtttcgtgt ctgtctctgt ctttgtgcgt gtttgtgccg
      721 gcatctaatg tttgcgcctg cgtctgtact agttagctaa ctagctctgt atctggcgga
      781 cccgtggtgg aactgacgag ttctgaacac ccggccgcaa ccctgggaga cgtcccaggg
      841 actttggggg ccgtttttgt ggcccgacct gaggaaggga gtcgatgtgg aatccgaccc
      901 cgtcaggata tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg
      961 aatttttgct ttcggtttgg aaccgaagcc gcgcgtcttg tctgctgcag cgctgcagca
     1021 tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga
     1081 ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc
     1141 acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct gcagaatggc
     1201 caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc atcacccagg
     1261 ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc ccctacatcg
     1321 tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt gtacacccta
     1381 agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct cctcgttcga
     1441 ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc ggaattcacg
     1501 cgtcaccatg aaaacatatg ctcctacatt attcatgttt ctatggctgc agctggatgg
     1561 gatgagtcga cgtaccagtt agatctgaga aatgtgactc cacccaaggt ctccttgttt
     1621 gagccatcaa aagcagagat tgcaaacaaa caaaaggcta ccctcgtgtg cttggccagg
     1681 ggcttcttcc ctgaccacgt ggagctgagc tggtgggtga atggcaagga ggtccacagt
     1741 ggggtcagca cggaccctca ggcctacaag gagagcaatt atagctactg cctgagcagc
     1801 cgcctgaggg tctctgctac cttctggcac aatcctcgca accacttccg ctgccaagtg
     1861 cagttccatg ggctttcaga ggaggacaag tggccagagg gctcacccaa acctgtcaca
     1921 cagaacatca gtgcagaggc ctggggccga gcagactgtg ggattacctc agcatcctat
     1981 caacaagggg tcttgtctgc caccatcctc tatgagatcc tgctagggaa agccaccctg
     2041 tatgctgtgc ttgtcagtac actggtggtg atggctatgg tcaaaagaaa gaactcatga
     2101 ctcgagcggg atcaattccg ccccccccct aacgttactg gccgaagccg cttggaataa
     2161 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     2221 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     2281 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
     2341 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     2401 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     2461 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     2521 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     2581 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     2641 aaccacgggg acgtggtttt cctttgaaaa acacgataat accatgaccg agtacaagcc
     2701 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     2761 gttcgccgac taccccgcca cgcgccacac cgtcgatcca gaccgccaca tcgagcgggt
     2821 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     2881 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     2941 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3001 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3061 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3121 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3181 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3241 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgactcgacc tgcaggcatg
     3301 caagcttcag gtagccggct aacgttaaca accggtacct ctagaactat agctagcatg
     3361 cgcaaattta aagcgctgat atcgataaaa taaaagattt tatttagtct ccagaaaaag
     3421 gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc
     3481 aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta ggaacagaga
     3541 gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg
     3601 gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag agaaccatca
     3661 gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa
     3721 tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc
     3781 acaacccctc actcggcgcg ccagtcctcc gatagactgc gtcgcccggg tacccgtgta
     3841 tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc
     3901 tcctctgagt gattgactac ccgtcagcgg gggtctttca tgggtaacag tttcttgaag
     3961 ttggagaaca acattctgag ggtaggagtc gaatattaag taatcctgac tcaattagcc
     4021 actgttttga atccacatac tccaatactc ctgaaatagt tcattatgga cagcgcagaa
     4081 agagctgggg agaattgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4141 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4201 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4261 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4321 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4381 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4441 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4501 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4561 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4621 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4681 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4741 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4801 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4861 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4921 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4981 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5041 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5101 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5161 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5221 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5281 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5341 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5401 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5461 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5521 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5581 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5641 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5701 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5761 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5821 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5881 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5941 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6001 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6061 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6121 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6181 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     6241 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     6301 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     6361 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     6421 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     6481 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
     6541 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     6601 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     6661 tttcccagtc acgacgttgt aaaacgacgg cgcaaggaat ggtgcatgca aggagatggc
     6721 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     6781 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     6841 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggcgattagt
     6901 ccaatttgtt aaagacagga tatcagtggt ccaggctcta gttttga
//
