LOCUS       sequence-436521-        6706 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector with Ametrine marker for expression shRNA with an 
            ""UltramiR"" microRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6706)
  AUTHORS   Ma EH, Dahabieh MS, DeCamp LM, Kaymak I, Kitchen-Goosen SM, Roy DG, 
            Verway MJ, Johnson RM, Samborska B, Scullion CA, Steadman M, Vos M, 
            Roddy TP, Krawczyk CM, Williams KS, Sheldon RD, Jones RG
  TITLE     (13)C metabolite tracing reveals glutamine and acetate as critical 
            in vivo fuels for CD8(+) T cells.
  JOURNAL   bioRxiv [Preprint]. 2023 Jun 11:2023.06.09.544407. doi: 
            10.1101/2023.06.09.544407.
  PUBMED    37333111
REFERENCE   2  (bases 1 to 6706)
  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: "bioRxiv 
            [Preprint]. 2023 Jun 11:2023.06.09.544407. doi: 
            10.1101/2023.06.09.544407."
COMMENT     Sequence Label: LMPd Ametrine Got2 shRNA#3
FEATURES             Location/Qualifiers
     source          1..6706
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           1443..1536
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1591..1605
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1632..1758
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        1772..2271
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1807..1830)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2204..2223
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(2292..2294,2295..2297,2298..3011)
                     /codon_start=1
                     /product="photostable yellow fluorescent protein with a 
                     large Stokes shift, excitable with violet light (Ai et al.,
                     2008)"
                     /label=mAmetrine1.1
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVRGEGEGDATNGKLTL
                     KFICTSGELPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GSYRTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNAWDAYITADKQKNGIK
                     ANFKIEHNVKDGGVQLADAYQQNTPIGDGSVLLPDNHYLSFQSKLFKDPNEQRDHMVLL
                     EFVTAAGITPGMDELYK"
     primer_bind     complement(2337..2358)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             3099..3613
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3775..3791
                     /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(3799..3805,3806..3823,3824..3829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3844..3865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3982..3999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4153..4741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4233..4252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4912..5703,5704..5772))
                     /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     5535..5554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5773..5877)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5945..5963
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6001..6023)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6123..6142
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6336..6358
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6486..6505
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6705..6706,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctctcga ctagggataa cagggtaatt
     1441 gtttgaatga ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc
     1501 tgggattact tcttcaggtt aacccaacag aaggctcgag aagtatattg ctgttgacag
     1561 tgagcgcgca gaccacatta cagaaattta gtgaagccac agatgtaaat ttctgtaatg
     1621 tggtctgctg cctactgcct cggaattcaa ggggctactt taggagcaat tatcttgttt
     1681 actaaaactg aataccttgc tatctctttg atacattttt acaaagctga attaaaatgg
     1741 tataaattaa atcacttttt tcaattctac cgggtagggg aggcgctttt cccaaggcag
     1801 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
     1861 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
     1921 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
     1981 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
     2041 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
     2101 ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg
     2161 ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa
     2221 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagc
     2281 ccaagcttac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     2341 tcgagctgga cggcgacgta aacggccaca ggttcagcgt gcgcggcgag ggcgagggcg
     2401 atgccaccaa cggcaagctg accctgaagt tcatctgcac ctccggcgag ctgcccgtgc
     2461 cctggcccac cctcgtgacc accctgtctt acggcgtgca gtgcttcgcc cgctaccccg
     2521 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2581 gcaccatctc cttcaaggac gacggcagct acaggacccg cgccgaggtg aagttcgagg
     2641 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2701 tcctggggca caagctggag tacaacatga acgcgtggga cgcgtatatc acggccgaca
     2761 agcagaagaa cggcatcaaa gcgaacttca agatcgagca caacgtcaag gacggcggcg
     2821 tgcagctcgc cgacgcgtac cagcagaaca cccccatcgg cgacggctcc gtgctgctgc
     2881 ctgacaacca ctacctgagc ttccagagca agctgttcaa agaccccaac gagcagcgcg
     2941 atcacatggt cctgctggag ttcgttaccg ccgccgggat aactcccggc atggacgagc
     3001 tgtacaagta agcggccgcg actctagagt cgacctgcag gcatgcaagc ttatcgataa
     3061 aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt
     3121 ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaatacat aactgagaat
     3181 agagaagttc agatcaaggt taggaacaga gagacagcag aatatgggcc aaacaggata
     3241 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg
     3301 tcccgccctc agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg
     3361 aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc
     3421 gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct
     3481 ccgatagact gcgtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc
     3541 gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc
     3601 gggggtcttt catgggtaac agtttcttga agttggagaa caacattctg agggtaggag
     3661 tcgaatatta agtaatcctg actcaattag ccactgtttt gaatccacat actccaatac
     3721 tcctgaaata gttcattatg gacagcgcag aaagagctgg ggagaattgt gaaattgtta
     3781 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3841 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3901 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3961 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4021 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4081 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4141 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4201 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4261 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4321 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4381 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4441 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4501 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4561 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4621 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4681 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4741 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4801 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4861 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4921 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4981 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5041 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5101 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5161 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5221 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5281 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5341 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5401 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5461 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5521 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5581 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5641 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5701 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5761 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5821 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5881 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     5941 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     6001 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     6061 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     6121 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     6181 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
     6241 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     6301 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     6361 ggcgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6421 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6481 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6541 gccacgatgc gtccggcgta gaggcgatta gtccaatttg ttaaagacag gatatcagtg
     6601 gtccaggctc tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca
     6661 tagataaaat aaaagatttt atttagtctc cagaaaaagg ggggaa
//
