LOCUS       sequence-237584-        6454 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Targeting vector for mouse Slc2a3 to insert 2A-tdiRFP670-PEST into 
            immediately upstream of stop codon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6454)
  AUTHORS   Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, 
            Nakano K, Saitoh N, Liu Z, Yamamoto T, Okamura T, Ohkawa Y, Kimura 
            H, Nikaido I
  TITLE     Genome-wide kinetic properties of transcriptional bursting in mouse 
            embryonic stem cells.
  JOURNAL   Sci Adv. 2020 Jun 17;6(25):eaaz6699. doi: 10.1126/sciadv.aaz6699. 
            eCollection 2020 Jun.
  PUBMED    32596448
REFERENCE   2  (bases 1 to 6454)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2020-06-17"; volume: "6(25):eaaz6699. doi"; pages: " 
            10.1126/sciadv.aaz6699. eCollection 2020 Jun"
COMMENT     Sequence Label: pTV-mSlc2a3-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..6454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1108..1161
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    complement(1162..1195)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1204..1224
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3187..3306
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3309..3342
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(4267..4287)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4267..4285)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4306..4322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4306..4322)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4319..4341)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4330..4346
                     /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(4354..4360,4361..4378,4379..4384))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4399..4420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4537..4554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4708..5296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4788..4807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5467..6258,6259..6327))
                     /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     6090..6109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6328..6418)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacccc
      661 acaggaagtc tttggaagca gctaattttt gtgactagaa aaaaaagaat ggcatatgac
      721 ttgcagagat acgtaatagc atcgtaagtc aactgaccaa tgatgctgtg gaagacttgt
      781 agcttaaggc tagtcagata gaaatgtgag gctcccctca catttatgaa cacttacatt
      841 ggtacatttg tatgaaacag tcgaccagca atgagcagtc ttctttctcc tgtctcctag
      901 gcctacttag gagcctacgt ttttatcatc ttcgctgcct tcctcatctt cttcctaatc
      961 ttcaccttct tcaaagtccc ggagaccaaa ggcaggactt tcgaggacat tgcccgggcc
     1021 ttcgaggggc aggcgcactc tggaaaaggc cctgccggtg tggagttgaa cagcatgcag
     1081 ccggtcaagg agacccctgg caacgccgag ggaagaggta gcctgctaac ttgcggggac
     1141 gtagaggaaa atcctggtcc cataacttcg tatagcatac attatacgaa gttatcaatg
     1201 ggcccaaaaa agaaaagaaa agttgaattc atggccagaa aagtggacct gaccagttgt
     1261 gaccgagagc cgatacacat tcctggctct atccagccat gtgggtgttt gctggcctgc
     1321 gatgcccagg cagtcaggat tactcgaatt accgaaaatg cgggtgcctt ctttggccgt
     1381 gaaacgcccc gtgtcggcga actcttagcc gattatttcg gagaaaccga ggctcacgca
     1441 cttaggaacg cactggcgca atcaagcgat cccaaacgcc ctgctctcat ctttgggtgg
     1501 agagatggac tgacaggccg tactttcgac ataagccttc accggcatga cggaaccagt
     1561 ataattgagt ttgaaccggc cgccgctgag caagcagaca acccccttcg cctgaccaga
     1621 caaattatcg cccgcactaa ggagctgaaa tcgctggaag aaatggccgc tcgtgttccc
     1681 aggtatcttc aggcaatgct gggttatcat agggtaatgc tctacagatt cgccgacgat
     1741 ggctctggta tggtaatagg ggaggccaag cgatccgacc tggagtcttt tctggggcag
     1801 catttccccg catctctagt accccagcag gcaaggcttt tgtacctcaa gaatgccatc
     1861 cgcgtggtat cggactcccg cggtattagt agcaggatcg ttccagaaca cgacgcctct
     1921 ggagctgcgc tggatctgtc tttcgctcac ctccgatcta tcagcccttg tcatttagag
     1981 tttctgcgaa atatgggggt cagcgcaagt atgtccctga gcataataat cgatggaaca
     2041 ctatggggcc taataatttg ccatcattac gaacctagag cagtaccaat ggcacagcga
     2101 gtggccgccg aaatgttcgc cgattttttg tctcttcatt ttactgccgc tcaccatcag
     2161 cgcggccacg ggactggctc gacaggttct ggcagtagtg ggactgcttc atcagaaaat
     2221 aataacatgg ccatggccag aaaagtggac ctgaccagtt gtgaccgaga gccgatacac
     2281 attcctggct ctatccagcc atgtgggtgt ttgctggcct gcgatgccca ggcagtcagg
     2341 attactcgaa ttaccgaaaa tgcgggtgcc ttctttggcc gtgaaacgcc ccgtgtcggc
     2401 gaactcttag ccgattattt cggagaaacc gaggctcacg cacttaggaa cgcactggcg
     2461 caatcaagcg atcccaaacg ccctgctctc atctttgggt ggagagatgg actgacaggc
     2521 cgtactttcg acataagcct tcaccggcat gacggaacca gtataattga gtttgaaccg
     2581 gccgccgctg agcaagcaga caaccccctt cgcctgacca gacaaattat cgcccgcact
     2641 aaggagctga aatcgctgga agaaatggcc gctcgtgttc ccaggtatct tcaggcaatg
     2701 ctgggttatc atagggtaat gctctacaga ttcgccgacg atggctctgg tatggtaata
     2761 ggggaggcca agcgatccga cctggagtct tttctggggc agcatttccc cgcatctcta
     2821 gtaccccagc aggcaaggct tttgtacctc aagaatgcca tccgcgtggt atcggactcc
     2881 cgcggtatta gtagcaggat cgttccagaa cacgacgcct ctggagctgc gctggatctg
     2941 tctttcgctc acctccgatc tatcagccct tgtcatttag agtttctgcg aaatatgggg
     3001 gtcagcgcaa gtatgtccct gagcataata atcgatggaa cactatgggg cctaataatt
     3061 tgccatcatt acgaacctag agcagtacca atggcacagc gagtggccgc cgaaatgttc
     3121 gccgattttt tgtctcttca ttttactgcc gctcaccatc agcgcggatc cgcgtttaaa
     3181 ctcgagtctc acggcttccc tcccgaggtg gaggagcagg ccgccggcac cctgcccatg
     3241 agctgcgccc aggagagcgg catggataga caccctgctg cttgcgccag cgccaggatc
     3301 aacgtcttga agttcctatt ctctagaaag tataggaact tctaggccgg gagcacctcc
     3361 ttcacctccc tccactgtgg aaagccacct ccccttaagt ggggagacct catcaggatg
     3421 aaccaggact gcttctgagt gctgctattc tcaccccaca cactccatga aaactcagct
     3481 gcacccaatg ctggggttga ccagatcgcc aatggctttt aagtgtttga tttgggggat
     3541 acttcccttg taatcaggaa agaccaagga agcctacctt tatattgggg gggaaggggc
     3601 cgcagctccc cttaggttct aaaacccgct aactaggaca actagggagt aggagtaggg
     3661 tgcagcaccg cccccaccac caccttagtt tgccaggaaa cagatcttca tacccagtgt
     3721 ggagggccct gggggattga acaaaagacc ccctcctcac ttgatacagc tctgcacagc
     3781 aaagtaactt gagttttatt tattttatct tcaggttgaa ttgcataaat atttattttt
     3841 ttaattgtaa ttttaccaaa taatgagaca gtaacgaaat tgaggttgga gggaggtgtt
     3901 taaagagagg ttcaggagga aagaaagttt ggtgctggag agaggttaca acagggagga
     3961 tggatgttac tggtggcctg tgagtcactt cctttgtaga tgaagggaca ctatcccttg
     4021 ttatttctca gaagagcgag tgcctgtact aaaaggctca tggctttctg tgggcctttt
     4081 cttttaagat tatagttttt atgtagtgtt actaaaatct aggcttgtta ctaaagtggt
     4141 ctttgtagtt agtgatattg atggattttt atgttgcatg gagtccagac aagggagggg
     4201 tcaccgcaag cctctccttt ccctggacca acaccctctc tgtcctgtga gctccagctt
     4261 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     4321 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4381 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4441 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4501 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4561 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4621 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4681 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4741 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4801 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4861 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4921 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4981 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5041 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5101 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5161 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5221 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5281 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5341 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5401 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5461 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5521 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5581 tggccccagt gctgcaatga taccgcggct gccacgctca ccggctccag atttatcagc
     5641 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5701 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5761 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5821 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5881 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5941 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6001 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6061 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6121 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6181 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6241 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6301 ggcgacacgg aaatgttgaa tactcatact cttccttttc aatattattg aagcatttat
     6361 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6421 gggattccgc gcacatttcc ccgaaaagtg ccac
//
