LOCUS       sequence-394840-        7771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low STIM1 abundance recombination plasmid for Matreyek Bxb1(GT) 
            landing pad in dual landing pad cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7771)
  AUTHORS   Roelle SM, Kamath ND, Matreyek KA
  TITLE     Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase 
            Sites for the Functional Characterization of Protein Variants.
  JOURNAL   ACS Synth Biol. 2023 Nov 3. doi: 10.1021/acssynbio.3c00355.
  PUBMED    37922210
REFERENCE   2  (bases 1 to 7771)
  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: "ACS Synth 
            Biol. 2023 Nov 3. doi: 10.1021/acssynbio.3c00355."
COMMENT     Sequence Label: 
            pNK005C_BattB_mkozak_STIM1_IRES_mCherry-H2A-P2A-PuroR
FEATURES             Location/Qualifiers
     source          1..7771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..40
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    join(2250..2824,2825..2827,2828..2836)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2417..2434)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2644..2663
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(2982..3000)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3250..3269
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3980..4036
                     /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     4530..4550
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4741..4862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4778..4797)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4832..4851
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(4871..4889)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4910..4926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4910..4926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4923..4945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4934..4950
                     /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(4958..4964,4965..4982,4983..4988))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5003..5024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5141..5158)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5312..5900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5392..5411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6071..6862,6863..6931))
                     /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     6694..6713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6932..7036)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7062..7517)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7199..7220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7411..7430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7644..7666
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7658..7675
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7659..7675
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7682..7701
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7682..7700
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccggcttgtc gacgacggcg gtctccgtcg tcaggatcat ccgcagatta taccgcaact
       61 acaccattgt atggatgtat gcgtccgtct tgccctgtgg ctcctctggg gactcctcct
      121 gcaccagggc cagagcctca gccatagtca cagtgagaag gcgacaggaa ccagctcggg
      181 ggccaactct gaggagtcca ctgcagcaga gttttgccga attgacaagc ccctgtgtca
      241 cagtgaggat gagaaactca gcttcgaggc agtccgtaac atccacaaac tgatggacga
      301 tgatgccaat ggtgatgtgg atgtggaaga aagtgatgag ttcctgaggg aagacctcaa
      361 ttaccatgac ccaacagtga aacacagcac cttccatggt gaggataagc tcatcagcgt
      421 ggaggacctg tggaaggcat ggaagtcatc agaagtatac aattggaccg tggatgaggt
      481 ggtacagtgg ctgatcacat atgtggagct gcctcagtat gaggagacct tccggaagct
      541 gcagctcagt ggccatgcca tgccaaggct ggctgtcacc aacaccacca tgacagggac
      601 tgtgctgaag atgacagacc ggagtcatcg gcagaagctg cagctgaagg ctctggacac
      661 tgtgctcttt gggcctcctc tcttgactcg ccataatcac ctcaaggact tcatgctggt
      721 ggtgtctatc gttattggtg tgggcggctg ctggtttgcc tatatccaga accgttactc
      781 caaggagcac atgaagaaga tgatgaagga cttggagggg ttacaccgag ctgagcagag
      841 tctgcatgac cttcaggaaa ggctgcacaa ggcccaggag gagcaccgca cagtggaggt
      901 ggagaaggtc catctggaaa agaagctgcg cgatgagatc aaccttgcta agcaggaagc
      961 ccagcggctg aaggagctgc gggagggtac tgagaatgag cggagccgcc aaaaatatgc
     1021 tgaggaggag ttggagcagg ttcgggaggc cttgaggaaa gcagagaagg agctagaatc
     1081 tcacagctca tggtatgctc cagaggccct tcagaagtgg ctgcagctga cacatgaggt
     1141 ggaggtgcaa tattacaaca tcaagaagca aaatgctgag aagcagctgc tggtggccaa
     1201 ggagggggct gagaagataa aaaagaagag aaacacactc tttggcacct tccacgtggc
     1261 ccacagctct tccctggatg atgtagatca taaaattcta acagctaagc aagcactgag
     1321 cgaggtgaca gcagcattgc gggagcgcct gcaccgctgg caacagatcg agatcctctg
     1381 tggcttccag attgtcaaca accctggcat ccactcactg gtggctgccc tcaacataga
     1441 ccccagctgg atgggcagta cacgccccaa ccctgctcac ttcatcatga ctgacgacgt
     1501 ggatgacatg gatgaggaga ttgtgtctcc cttgtccatg cagtccccta gcctgcagag
     1561 cagtgttcgg cagcgcctga cggagccaca gcatggcctg ggatctcaga gggatttgac
     1621 ccattccgat tcggagtcct ccctccacat gagtgaccgc cagcgtgtgg cccccaaacc
     1681 tcctcagatg agccgtgctg cagacgaggc tctcaatgcc atgacttcca atggcagcca
     1741 ccggctgatc gagggggtcc acccagggtc tctggtggag aaactgcctg acagccctgc
     1801 cctggccaag aaggcattac tggcgctgaa ccatgggctg gacaaggccc acagcctgat
     1861 ggagctgagc ccctcagccc cacctggtgg ctctccacat ttggattctt cccgttctca
     1921 cagccccagc tccccagacc cagacacacc atctccagtt ggggacagcc gagccctgca
     1981 agccagccga aacacacgca ttccccacct ggctggcaag aaggctgtgg ctgaggagga
     2041 taatggctct attggcgagg aaacagactc cagcccaggc cggaagaagt ttcccctcaa
     2101 aatctttaag aagcctctta agaagtagta acttaagaat tcaccggtct gacctccttc
     2161 tccctctctt caggtctgca gtcgacggta ccgcgggccc gggatccacc ggatctagat
     2221 aactgatcac tgtggaatca gagagactgc ccctctccct ctcccccccc taacgttact
     2281 ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata
     2341 ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt
     2401 cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa
     2461 gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag
     2521 cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca
     2581 cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc
     2641 aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat
     2701 tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta
     2761 aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga
     2821 taatatggcc acaaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
     2881 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     2941 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     3001 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     3061 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     3121 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     3181 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     3241 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     3301 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     3361 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     3421 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     3481 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggctact cagatctcga
     3541 gctcaagctt cgaattctga tgtcgggacg cggcaagcag ggaggcaaag ctcgcgccaa
     3601 agccaagacc cgctcttctc gtgccggtct ccagttcccc gtgggccgag tgcaccgact
     3661 gctccgcaag ggcaactatg ctgagcgggt cggggccggc gcgccggtgt acctggcggc
     3721 ggtgctggag tacctgactg ccgagatcct ggagctggcg ggcaacgccg cccgcgacaa
     3781 caagaagacc cgcattatcc cgcgccactt gcagctggcc atccgcaacg acgaggagct
     3841 caacaagctg ctgggcaaag taaccatcgc tcagggtggt gtcctgccca acatccaggc
     3901 tgtgctactg cccaagaaga ccgagagtca ccacaaggcc aaaggcaaac gggatccacc
     3961 ggatctagat aacagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga
     4021 ggagaaccct ggacctaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     4081 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     4141 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     4201 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     4261 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     4321 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     4381 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     4441 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     4501 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     4561 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     4621 gtaataggga tcctgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     4681 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     4741 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4801 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4861 taggagatcc cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt
     4921 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4981 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5041 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5101 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5161 actcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5221 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5281 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5341 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5401 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5461 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5521 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5581 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5641 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5701 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5761 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5821 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5881 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5941 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6001 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6061 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6121 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6181 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6241 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6301 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6361 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6421 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6481 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6541 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6601 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6661 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6721 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6781 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6841 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6901 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6961 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7021 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     7081 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     7141 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     7201 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     7261 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     7321 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     7381 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     7441 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     7501 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     7561 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     7621 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
     7681 gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg tcgacgatgt
     7741 aggtcacggc attccggact cagcgctcga g
//
