LOCUS       sequence-324900-        5463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and mEGFP-linker sequence sequence for N-terminus 
            tagging of human POLR2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5463)
  TITLE     Allen Institute for Cell Sciences Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5463)
  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: 
            "Unpublished"
COMMENT     Sequence Label: AICSDP-117:POLR2A-mEGFP
FEATURES             Location/Qualifiers
     source          1..5463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(1035..1037,1038..1040,1041..1751)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1080..1101)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1341..1360)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1688..1709
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2864..2880)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2864..2880)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2877..2899)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2888..2904
                     /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(2912..2918,2919..2936,2937..2942))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2957..2978
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3095..3112)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3266..3854)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3346..3365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4025..4816,4817..4885))
                     /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     4648..4667
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4886..4990)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5058..5076
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5114..5136)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5236..5255
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(5449..5463,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(5463,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgacgcgta ttgggataag tctcttcgag aggctcaggc
       61 catggatcta ggatcatggc tcggtacagc gcggcattac aggccaggag atgcccaccc
      121 gtctccgggg gccgtttgac aaacatcggc tacttccgga caagccactg gtcctgccat
      181 cttggtttgt tcatgcatat tcagcaagag aactgcatat tcatgagcaa ccgcccttcc
      241 ccgggaggac ccgccccgcc ccgccgccat taccgattcg cagagcagac cattttccag
      301 ctgggggggg ctacgaatga gaggcttttg ctgaagatga aaccgttgtc caaactagaa
      361 tgaaagcggc ccaattttac cctcgagtct tcaaacctta agcctcagtg caaaagggag
      421 cagcgaggag aggcggtggg attaaagaaa aaaattcttc tctccctgtc acttcaaaag
      481 gtcgcccaac cagcgagcgg ggggtttacc cacgactctg gctcgcaccc cttgcgggcc
      541 tgcgcaggcg gccccgtagc gcaagggagg gcgggaaagg aaggggcggg gacgcgaggg
      601 cgaatctata agaagcgtcg ttcagcgagt tcgctgctca gaagcgccga gagcgcggcc
      661 gggacggttg gagaagaagg cggctcccgg aagggggaga gacaaactgc cgtaacctct
      721 gccgttcagg aacccggtta cttatttatt cgttaccctt tttcttcttc ctcccccaaa
      781 aaccttttcc ttttcccttc tttttttttc ctttttggga gctgaaaaat ttccggtaag
      841 ggaaagaagg gctcctttcg ctccttattt ccccgcctct ttccctcccc caccttcccc
      901 tcctccggct ttttcctccc aactcgggga ggtccttccc ggtggccgcc ctgacgaggt
      961 ctgagcacct aggcggaggc ggcgcaggct ttttgtagtg aggtttgcgc ctgcgcagcg
     1021 cgcctgcctc cgccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1081 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1141 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1201 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     1261 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1321 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1381 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1441 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1501 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1561 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1621 tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc
     1681 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1741 agctgtacaa gagcggcctg agaagcagag cccaggcctc taactccgcc gtcgacggca
     1801 ccgctggacc cggaagcaca ggcagctccc acgggggtgg acccccctcg ggggacagcg
     1861 catgcccgct gcgcaccatc aagagagtcc agttcggagt cctgagtccg gatgaactgg
     1921 taagcggctc tgtcctcccc ttcccccctc ctcccctggc gggcggggcc ggacgggggc
     1981 tgcggaaact tggcgctttc tcgctgctta tgggtgacgg gccaggagca tggctcagca
     2041 gcgccaaggc cgtgtaggcg ccagtctcgg gcctcccaga gttatatttt gcaaagagtt
     2101 gagcaggcgt agcgctttgt ccgagatggg gggctggctg gagggtgaga aggagggaga
     2161 gaaggatgac tgatttcaca ctccagagtt accgacgtta aaggcgatcc gacgaatcct
     2221 agggagtctt tacaagtttg gttaagaagc caaacccggg tagcttcctc ctcttctgta
     2281 atacaaatgt taactgagca cctgctctgt tgaagacaca ggagactcgg gttacagagg
     2341 tgaacacgac agaccaggac cctaccttca agagcactgt ctgtgggaaa gggcaagcag
     2401 taaacatgaa tacaatgaat gaatgagata attcagttaa tggcatttgt ctcgaataga
     2461 gtgaaaaggg gtaatcccat ggagagtcac tgagctgggg ctactttatt tttttatttt
     2521 tattttattt tttgagacgg agtcttgctc tgtcgcccag gctggagtgc aatggcacga
     2581 tctcggctca ctgcaatctc cgcctcccgg gttcaagcga ttctcctgcc ttagcctcct
     2641 gagtagctgg gattacaggc atgcgccacc atgccccgct aatttttgta tttttagtag
     2701 aggaggggtt ttgccatgtt ggccaggctg gtctcgaacg cctgccctga ggtgattctc
     2761 ccccgtcttt tttttttttt tttttttttt gagacggagt ttcgctctta ttccccaggc
     2821 cagagtgcaa tcccaatggc gcgccgagct tggcgtaatc atggtcatag ctgtttcctg
     2881 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     2941 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3001 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3061 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3121 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3181 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3241 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3301 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3361 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3421 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3481 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3541 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3601 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3661 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3721 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3781 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3841 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3901 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3961 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4021 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4081 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4141 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4201 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4261 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4321 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4381 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4441 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4501 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4561 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4621 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4681 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4741 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4801 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4861 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4921 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4981 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     5041 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     5101 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     5161 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     5221 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     5281 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc
     5341 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     5401 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     5461 gtt
//
