LOCUS       sequence-379964-        5457 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Knock in HA-T2A-mCherry into the mouse Tmsb10 gene locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5457)
  TITLE     Tmsb10 triggers fetal Leydig differentiation by suppressing the 
            RAS/ERK pathway.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5457)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKnockinDonor-HA-mCherry-T2A-Tmsb10
FEATURES             Location/Qualifiers
     source          1..5457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(32..460)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(142..163)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     354..373
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     587..609
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     601..618
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     602..618
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     628..647
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        628..646
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1534..1560
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1588..2295
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     2001..2020
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2296..2349
                     /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"
     primer_bind     complement(3218..3234)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3218..3234)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3271..3291)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3271..3289)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3310..3326)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3310..3326)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3323..3345)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3334..3350
                     /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(3358..3364,3365..3382,3383..3388))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3403..3424
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3541..3558)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3712..4300)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3792..3811)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4471..5262,5263..5331))
                     /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     5094..5113
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5332..5436)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
       61 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
      121 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
      181 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
      241 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
      301 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
      361 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
      421 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
      481 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
      541 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
      601 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc
      661 gggccccccc tcgaggtcga cagaaggaga ctctggagag caaacgcaag agacttgagg
      721 cgacgcgggt ggggtgtctc ccgcgagcag ggctgcagca gagcgcgcgt gctcgcccgc
      781 acacccacaa ccctacacca cacatacatt caacaaaacc ccaagtcctt ccccgctgcc
      841 ccccccccca cgcccttccc gcggggtgag cagcaggctc ctgggtacta ggaaaaggag
      901 atatatcaag aaaagagacc accgggagct ggtgcctgcc accctccacg cgggatcgca
      961 acgggttagg aacgcgggcc gagaagggcg gagccccagg cagcctggag atgctgagcg
     1021 ccccccagcc cccgcgcgcg ctggccacgc cctccgcgcg ggctggccac gccctcgttg
     1081 cggcgggcga gaggcggggc ggggcgcggt gtatataaga cgaggggcgg gcgccgctct
     1141 tttgtctctt gctgcagcaa cgagagtggg agcacctgga gcgcgagctc ggaaggagaa
     1201 tccacggtaa ggcacagcgc cggccttggg gcctaccctg ggtggggtcg ggccagttta
     1261 tggctggcgg gcgcgttccc cttcggttgc aacaggcgac ccggcggttt ggaatcttct
     1321 ggggctagca ggccacgccc tggggcttcg gagggagggt gtggtggacg agtctaggat
     1381 cctcggcctc cgggctgcgg gaacgcccag gactgggaat gccacgttga gaggcgttcg
     1441 cggaaggcgc gggatccagg acgcgctggt cacccccaag cccctggcca cccattatca
     1501 ccttgtttcg cccatcagag ttgtaagaaa atgtacccat acgatgttcc agattacgct
     1561 cttatggcca tggaggcccg aattcggatg gtgagcaagg gcgaggagga taacatggcc
     1621 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     1681 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     1741 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     1801 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     1861 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1921 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1981 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     2041 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     2101 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     2161 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     2221 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     2281 gacgagctgt acaaggaggg cagaggcagt ctgctgacat gcggtgacgt ggaagagaat
     2341 cccggcccta tggcagacaa gccggacatg ggggaaatcg ccagcttcga taaggccaag
     2401 ctgaagaaaa ccgagacgca ggagaagaac accctgccga ccaaagagag taagtgtgcc
     2461 ccgggttccc accccagcgc tcctcccttc cctgctgtgc cagcccggca cccccacccc
     2521 catccggcca ctatcttccc accccgaatc taaggccaca gccacccttc cagtcacaca
     2581 aagcactttg tctcttccaa accccttctc tgtccgcagg cctcgtgggt gcctcaccca
     2641 caccaggttt gtgggttggg tatggggggc tgtaaggtag ctccaatgac cacatccctc
     2701 ccttcgcagc cattgaacag gaaaagagga gtgaaatctc ctaaaagcct aggaagattt
     2761 ccccacccca ccccaccccg ccccatcatc tccaagaccc cctcgtgatg tggaggaaga
     2821 gccacctgca agatggacgc gagccacaag ctgcactgtg aaacccgggc actccgagcc
     2881 gatgccaccg gcccgcgggt ctctgaaggg gacccctcca ctaatcggac tgccaaattt
     2941 caccggtttg ccctgggata ttatagaaaa ttatttgtat gattgatgaa aataaaaaca
     3001 cctcgtggca tggttggcgt ggtctgaatt ttttagttga gtatgcgtgg gcagagcggt
     3061 ctgggagtga gttgtgccgc agggagagag gaggggacgt ggtagaatgg gtgaggagga
     3121 tgtatcgttt ttatttttgt tttacgtgct tctcgacatg cggtttcctg ggttctcagt
     3181 tgcttcgccc ttaatgcgga attcctgcag cccgggggat ccactagttc tagagcggcc
     3241 gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     3301 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3361 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3421 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3481 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3541 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3601 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3661 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3721 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3781 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3841 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3901 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3961 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4021 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4081 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4141 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4201 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4261 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4321 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4381 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4441 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4501 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4561 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4621 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4681 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4741 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4801 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4861 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4921 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4981 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5041 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5101 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5161 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5221 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5281 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5341 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5401 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgcc
//
