LOCUS       sequence-436524-        6708 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 6708)
  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 6708)
  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 Got1 shRNA#1
FEATURES             Location/Qualifiers
     source          1..6708
                     /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..1564
                     /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           1592..1606
                     /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           1634..1760
                     /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        1774..2273
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1809..1832)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2206..2225
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(2294..2296,2297..2299,2300..3013)
                     /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(2339..2360)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             3101..3615
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3777..3793
                     /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(3801..3807,3808..3825,3826..3831))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3846..3867
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3984..4001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4155..4743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4235..4254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4914..5705,5706..5774))
                     /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     5537..5556
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5775..5879)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5947..5965
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6003..6025)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6125..6144
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6338..6360
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6488..6507
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6707..6708,1..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 aaggtatatt gctgttgaca
     1561 gtgagcgcgc agaagattgc taatgacaat agtgaagcca cagatgtatt gtcattagca
     1621 atcttctgct tgcctactgc ctcggaattc aaggggctac tttaggagca attatcttgt
     1681 ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat
     1741 ggtataaatt aaatcacttt tttcaattct accgggtagg ggaggcgctt ttcccaaggc
     1801 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     1861 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     1921 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     1981 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     2041 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     2101 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     2161 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     2221 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     2281 gcccaagctt accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2341 ggtcgagctg gacggcgacg taaacggcca caggttcagc gtgcgcggcg agggcgaggg
     2401 cgatgccacc aacggcaagc tgaccctgaa gttcatctgc acctccggcg agctgcccgt
     2461 gccctggccc accctcgtga ccaccctgtc ttacggcgtg cagtgcttcg cccgctaccc
     2521 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2581 gcgcaccatc tccttcaagg acgacggcag ctacaggacc cgcgccgagg tgaagttcga
     2641 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2701 catcctgggg cacaagctgg agtacaacat gaacgcgtgg gacgcgtata tcacggccga
     2761 caagcagaag aacggcatca aagcgaactt caagatcgag cacaacgtca aggacggcgg
     2821 cgtgcagctc gccgacgcgt accagcagaa cacccccatc ggcgacggct ccgtgctgct
     2881 gcctgacaac cactacctga gcttccagag caagctgttc aaagacccca acgagcagcg
     2941 cgatcacatg gtcctgctgg agttcgttac cgccgccggg ataactcccg gcatggacga
     3001 gctgtacaag taagcggccg cgactctaga gtcgacctgc aggcatgcaa gcttatcgat
     3061 aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag
     3121 gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaatac ataactgaga
     3181 atagagaagt tcagatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga
     3241 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc
     3301 ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc
     3361 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
     3421 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
     3481 ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
     3541 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
     3601 gcgggggtct ttcatgggta acagtttctt gaagttggag aacaacattc tgagggtagg
     3661 agtcgaatat taagtaatcc tgactcaatt agccactgtt ttgaatccac atactccaat
     3721 actcctgaaa tagttcatta tggacagcgc agaaagagct ggggagaatt gtgaaattgt
     3781 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3841 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3901 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3961 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4021 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4081 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4141 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4201 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4261 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4321 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4381 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4441 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4501 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4561 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4621 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4681 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4741 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4801 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4861 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4921 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4981 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5041 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5101 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5161 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5221 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5281 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5341 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5401 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5461 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5521 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5581 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5641 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5701 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5761 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5821 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5881 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     5941 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     6001 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     6061 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     6121 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     6181 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
     6241 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     6301 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     6361 acggcgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6421 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6481 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6541 cggccacgat gcgtccggcg tagaggcgat tagtccaatt tgttaaagac aggatatcag
     6601 tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata gagtacgagc
     6661 catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaa
//
