LOCUS       sequence-209766-        5403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for C-terminus tagging of 
            human GJA1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5403)
  AUTHORS   Allen Institute for Cell Science
  TITLE     Allen Institute for Cell Science Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5403)
  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            "Unpublished"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-43: GJA1-mEGFP
FEATURES             Location/Qualifiers
     source          1..5403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     179..201
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     193..210
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     194..210
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(1249..1251,1252..1965)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1291..1312)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1552..1571)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1899..1920
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2997..3013)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2997..3013)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3010..3032)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3021..3037
                     /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(3045..3051,3052..3069,3070..3075))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3090..3111
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3228..3245)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3399..3987)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3479..3498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4158..4949,4950..5018))
                     /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     4781..4800
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5019..5123)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5191..5209
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5247..5269)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5369..5388
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg
       61 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
      121 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
      181 cagtcacgac gttgtaaaac gacggccagt gaattgacgc gtattgggat gtctgccttt
      241 cgttgtaaca ctcagcaacc tggttgtgaa aatgtctgct atgacaagtc tttcccaatc
      301 tctcatgtgc gcttctgggt cctgcagatc atatttgtgt ctgtacccac actcttgtac
      361 ctggctcatg tgttctatgt gatgcgaaag gaagagaaac tgaacaagaa agaggaagaa
      421 ctcaaggttg cccaaactga tggtgtcaat gtggacatgc acttgaagca gattgagata
      481 aagaagttca agtacggtat tgaagagcat ggtaaggtga aaatgcgagg ggggttgctg
      541 cgaacctaca tcatcagtat cctcttcaag tctatctttg aggtggcctt cttgctgatc
      601 cagtggtaca tctatggatt cagcttgagt gctgtttaca cttgcaaaag agatccctgc
      661 ccacatcagg tggactgttt cctctctcgc cccacggaga aaaccatctt catcatcttc
      721 atgctggtgg tgtccttggt gtccctggcc ttgaatatca ttgaactctt ctatgttttc
      781 ttcaagggcg ttaaggatcg ggttaaggga aagagcgacc cttaccatgc gaccagtggt
      841 gcgctgagcc ctgccaaaga ctgtgggtct caaaaatatg cttatttcaa tggctgctcc
      901 tcaccaaccg ctcccctctc gcctatgtct cctcctgggt acaagctggt tactggcgac
      961 agaaacaatt cttcttgccg caattacaac aagcaagcaa gtgagcaaaa ctgggctaat
     1021 tacagtgcag aacaaaatcg aatggggcag gcgggaagca ccatctctaa ctcccatgca
     1081 cagccttttg atttccccga tgataaccag aattctaaaa aactagctgc tggacatgaa
     1141 ttacagccac tagccattgt ggaccagcga ccttcaagca gagccagcag tcgtgccagc
     1201 agcagacctc gcccagacga cctggagatc gaccctcctg tggccacagt gagcaagggc
     1261 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1321 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1381 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1441 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1501 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1561 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1621 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1681 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1741 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1801 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1861 tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1921 accgccgccg ggatcactct cggcatggac gagctgtaca agtagataca ggcttgaaag
     1981 catcaagatt ccactcaatt gtgcagaaga aaaaaggtgc tgtagaaagt gcaccaggtg
     2041 ttaattttga tccggtggag gtggtactca acagccttat tcatgaggct tagaaaacac
     2101 aaagacatta gaatacctag gttcactggg ggtgtatggg gtagatgggt ggagagggag
     2161 gggataagag aggtgcatgt tggtatttaa agtagtggat tcaaagaact tagattataa
     2221 ataagagttc cattaggtga tacatagata agggcttttt ctccccgcaa acacccctaa
     2281 gaatggttct gtgtatgtga atgagcgggt ggtaattgtg gctaaatatt tttgttttac
     2341 caagaaactg aaataattct ggccaggaat aaatacttcc tgaacatctt aggtcttttc
     2401 aacaagaaaa agacagagga ttgtccttaa gtccctgcta aaacattcca ttgttaaaat
     2461 ttgcactttg aaggtaagct ttctaggcct gaccctccag gtgtcaatgg acttgtgcta
     2521 ctatattttt ttattcttgg tatcagttta aaattcagac aaggcccaca gaataagatt
     2581 ttccatgcat ttgcaaatac gtatattctt tttccatcca cttgcacaat atcattacca
     2641 tcactttttc atcattcctc agctactact cacattcatt taatggtttc tgtaaacatt
     2701 tttaagacag ttgggatgtc acttaacatt ttttttttga gctaaagtca gggaatcaag
     2761 ccatgcttaa tatttaacaa tcacttatat gtgtgtcgaa gagtttgttt tgtttgtcat
     2821 gtattggtac aagcagatac agtataaact cacaaacaca gatttgaaaa taatgcacat
     2881 atggtgttca aatttgaacc tttctcatgg atttttgtgg tgtgggccaa tatggtgttt
     2941 acattatata attcctgctg tgatcccaat ggcgcgccga gcttggcgta atcatggtca
     3001 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3061 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3121 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3181 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3241 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3301 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3361 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3421 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3481 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3541 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3601 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3661 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3721 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3781 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3841 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3901 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3961 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4021 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4081 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4141 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4201 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4261 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4321 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4381 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4441 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4501 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4561 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4621 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4681 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4741 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4801 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4861 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4921 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4981 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5041 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5101 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     5161 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     5221 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     5281 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     5341 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     5401 cat
//
