LOCUS       sequence-461350-        8488 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Low expression plasmid of STIM1 with N-terminal mGreenLantern tag 
            for Matreyek Bxb1 (GT) landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8488)
  TITLE     Comprehensive mutational characterization of the calcium-sensing 
            STIM1 EF-hand reveals residues essential for structure and function
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8488)
  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            "Unpublished"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: 
            pNK036C_AttB_mkozak_mGreenLantern-STIM1_IRES_mCherry-H2A-P2A-PuroR
FEATURES             Location/Qualifiers
     source          1..8488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(113..217)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(243..698)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(380..401)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     592..611
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     825..847
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     839..856
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     840..856
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    955..992
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             join(1140..1142,1143..1145,1146..1856)
                     /codon_start=1
                     /product="monomeric GFP derivative that displays 
                     exceptionally high brightness in cells (Campbell et al., 
                     2020)"
                     /label=mGreenLantern
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIVLKGIDFKEDGNILGHKLEYNFNSHKVYITADKQKNGIK
                     ANFKTRHNVEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLK
                     ERVTAAGITHDMDELYK"
     primer_bind     complement(1185..1206)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    join(3919..4493,4494..4496,4497..4505)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4086..4103)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4313..4332
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(4651..4669)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4919..4938
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5649..5705
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     6199..6219
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6410..6531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6447..6466)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6501..6520
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(6540..6558)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6579..6595)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6579..6595)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6592..6614)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6603..6619
                     /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(6627..6633,6634..6651,6652..6657))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6672..6693
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6810..6827)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6981..7569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7061..7080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7740..8488,1..43,44..112))
                     /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     8363..8382
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
       61 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
      121 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      181 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
      241 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
      301 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      361 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      421 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      481 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      541 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      601 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      661 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      721 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      781 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      841 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccgggcc
      901 ccccctcgag gtcgacgatg taggtcacgg cattccggac tcagcgctcg agccggcttg
      961 tcgacgacgg cggtctccgt cgtcaggatc atccgcagat tataccgcaa ctacaccatt
     1021 gtatggatgt atgcgtccgt cttgccctgt ggctcctctg gggactcctc ctgcaccagg
     1081 gccagagcct cagccatagt cacagtgaga aggcgacagg aaccagctcg ggggccaaca
     1141 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1201 gcgacgtaaa cggccacaag ttcagcgtcc gcggcgaggg cgagggcgat gccaccaacg
     1261 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1321 tcgtgaccac cttaggctac ggcgtggcct gcttcgcccg ctaccccgac cacatgaagc
     1381 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatctctt
     1441 tcaaggacga cggtacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1501 tgaaccgcat cgtgctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1561 agctggagta caacttcaac agccacaagg tctatatcac ggccgacaag cagaagaacg
     1621 gcatcaaggc taacttcaag acccgccaca acgttgagga cggcggcgtg cagctcgccg
     1681 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1741 acctgagcca tcagtccaaa ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1801 tgaaggagag ggtgaccgcc gccgggatta cacatgacat ggacgagctg tacaagtctg
     1861 aggagtccac tgcagcagag ttttgccgaa ttgacaagcc cctgtgtcac agtgaggatg
     1921 agaaactcag cttcgaggca gtccgtaaca tccacaaact gatggacgat gatgccaatg
     1981 gtgatgtgga tgtggaagaa agtgatgagt tcctgaggga agacctcaat taccatgacc
     2041 caacagtgaa acacagcacc ttccatggtg aggataagct catcagcgtg gaggacctgt
     2101 ggaaggcatg gaagtcatca gaagtataca attggaccgt ggatgaggtg gtacagtggc
     2161 tgatcacata tgtggagctg cctcagtatg aggagacctt ccggaagctg cagctcagtg
     2221 gccatgccat gccaaggctg gctgtcacca acaccaccat gacagggact gtgctgaaga
     2281 tgacagaccg gagtcatcgg cagaagctgc agctgaaggc tctggacact gtgctctttg
     2341 ggcctcctct cttgactcgc cataatcacc tcaaggactt catgctggtg gtgtctatcg
     2401 ttattggtgt gggcggctgc tggtttgcct atatccagaa ccgttactcc aaggagcaca
     2461 tgaagaagat gatgaaggac ttggaggggt tacaccgagc tgagcagagt ctgcatgacc
     2521 ttcaggaaag gctgcacaag gcccaggagg agcaccgcac agtggaggtg gagaaggtcc
     2581 atctggaaaa gaagctgcgc gatgagatca accttgctaa gcaggaagcc cagcggctga
     2641 aggagctgcg ggagggtact gagaatgagc ggagccgcca aaaatatgct gaggaggagt
     2701 tggagcaggt tcgggaggcc ttgaggaaag cagagaagga gctagaatct cacagctcat
     2761 ggtatgctcc agaggccctt cagaagtggc tgcagctgac acatgaggtg gaggtgcaat
     2821 attacaacat caagaagcaa aatgctgaga agcagctgct ggtggccaag gagggggctg
     2881 agaagataaa aaagaagaga aacacactct ttggcacctt ccacgtggcc cacagctctt
     2941 ccctggatga tgtagatcat aaaattctaa cagctaagca agcactgagc gaggtgacag
     3001 cagcattgcg ggagcgcctg caccgctggc aacagatcga gatcctctgt ggcttccaga
     3061 ttgtcaacaa ccctggcatc cactcactgg tggctgccct caacatagac cccagctgga
     3121 tgggcagtac acgccccaac cctgctcact tcatcatgac tgacgacgtg gatgacatgg
     3181 atgaggagat tgtgtctccc ttgtccatgc agtcccctag cctgcagagc agtgttcggc
     3241 agcgcctgac ggagccacag catggcctgg gatctcagag ggatttgacc cattccgatt
     3301 cggagtcctc cctccacatg agtgaccgcc agcgtgtggc ccccaaacct cctcagatga
     3361 gccgtgctgc agacgaggct ctcaatgcca tgacttccaa tggcagccac cggctgatcg
     3421 agggggtcca cccagggtct ctggtggaga aactgcctga cagccctgcc ctggccaaga
     3481 aggcattact ggcgctgaac catgggctgg acaaggccca cagcctgatg gagctgagcc
     3541 cctcagcccc acctggtggc tctccacatt tggattcttc ccgttctcac agccccagct
     3601 ccccagaccc agacacacca tctccagttg gggacagccg agccctgcaa gccagccgaa
     3661 acacacgcat tccccacctg gctggcaaga aggctgtggc tgaggaggat aatggctcta
     3721 ttggcgagga aacagactcc agcccaggcc ggaagaagtt tcccctcaaa atctttaaga
     3781 agcctcttaa gaagtagtaa cttaagaatt caccggtctg acctccttct ccctctcttc
     3841 aggtctgcag tcgacggtac cgcgggcccg ggatccaccg gatctagata actgatcact
     3901 gtggaatcag agagactgcc cctctccctc tcccccccct aacgttactg gccgaagccg
     3961 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt
     4021 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
     4081 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
     4141 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
     4201 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
     4261 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc
     4321 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
     4381 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct
     4441 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca
     4501 caaccatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     4561 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     4621 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     4681 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     4741 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     4801 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     4861 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     4921 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     4981 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     5041 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     5101 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     5161 acgaacgcgc cgagggccgc cactccaccg gcggctactc agatctcgag ctcaagcttc
     5221 gaattctgat gtcgggacgc ggcaagcagg gaggcaaagc tcgcgccaaa gccaagaccc
     5281 gctcttctcg tgccggtctc cagttccccg tgggccgagt gcaccgactg ctccgcaagg
     5341 gcaactatgc tgagcgggtc ggggccggcg cgccggtgta cctggcggcg gtgctggagt
     5401 acctgactgc cgagatcctg gagctggcgg gcaacgccgc ccgcgacaac aagaagaccc
     5461 gcattatccc gcgccacttg cagctggcca tccgcaacga cgaggagctc aacaagctgc
     5521 tgggcaaagt aaccatcgct cagggtggtg tcctgcccaa catccaggct gtgctactgc
     5581 ccaagaagac cgagagtcac cacaaggcca aaggcaaacg ggatccaccg gatctagata
     5641 acagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg
     5701 gacctaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     5761 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     5821 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     5881 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     5941 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     6001 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     6061 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     6121 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     6181 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     6241 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag taatagggat
     6301 cctgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     6361 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     6421 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     6481 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aggagatccc
     6541 ctttagtgag ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga
     6601 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     6661 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     6721 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6781 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgca ctcggtcgtt
     6841 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6901 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6961 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     7021 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     7081 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     7141 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     7201 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     7261 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     7321 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     7381 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     7441 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     7501 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     7561 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     7621 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     7681 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     7741 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7801 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7861 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7921 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7981 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     8041 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     8101 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     8161 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     8221 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     8281 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     8341 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     8401 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     8461 agttcgatgt aacccactcg tgcaccca
//
