LOCUS       sequence-404715-        6745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  jGCaMP7c recombination plasmid for Matreyek Bxb1(GA) landing pad in 
            dual landing pad cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6745)
  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 6745)
  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: G1095C_BattB(GA)_jGCaMP7c_IRES_shBleR-P2A-HygroR
FEATURES             Location/Qualifiers
     source          1..6745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     621..640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(859..963)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(989..1444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1126..1147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1338..1357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1571..1593
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1585..1602
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1586..1602
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1609..1628
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1609..1627
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1701..1738
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      1763..1772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1769..1777,1778..1795,1796..1798,1799..1831,1832..1834,
                     1835..1858,1859..1885,1886..1942,1943..1945,1946..1948,
                     1949..2218,2219..2236,2237..2671,2672..2677,2678..3121)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6m
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSQPNVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKGSYRDSEEEIREAFGVFDKDGN
                     GYISAAELRHVMNNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1865..1874
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2155..2176
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2282..2303)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2543..2562)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(3243..3817,3818..3820,3821..3829)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3410..3427)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3637..3656
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3830..4201
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     CDS             4208..4264
                     /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"
     CDS             4277..5296
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             4277..5296
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     polyA_signal    5413..5534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5450..5469)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5504..5523
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(5543..5561)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5582..5598)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5582..5598)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5595..5617)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5606..5622
                     /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(5630..5636,5637..5654,5655..5660))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5675..5696
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5813..5830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5984..6572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6064..6083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6743..6745,1..789,790..858))
                     /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"
ORIGIN
        1 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
       61 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      121 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      181 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
      241 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      301 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      361 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      421 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
      481 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
      541 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      601 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      661 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      721 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      781 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      841 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      901 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      961 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     1021 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1081 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1141 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1201 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1261 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     1321 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     1381 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     1441 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     1501 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     1561 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca
     1621 ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acgatgtagg tcacggcatt
     1681 ccggactcag cgctcgagcc ggcttgtcga cgacggcgga ctccgtcgtc aggatcatcc
     1741 gcagattata ccgcaactac acgccaccat gggttctcat catcatcatc atcatggtat
     1801 ggctagcatg actggtggac agcaaatggg tcgggatctg tacgacgatg acgataagga
     1861 tctcgccacc atggtcgact catcacgtcg taagtggaat aagacaggtc acgcagtcag
     1921 agctataggt cggctgagct cacagcctaa cgtctatatc aaggccgaca agcagaagaa
     1981 cggcatcaag gcgaacttca agatccgcca caacatcgag gacggcggcg tgcagctcgc
     2041 ctaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2101 ctacctgagc gtgcagtcca aactttcgaa agaccccaac gagaagcgcg atcacatggt
     2161 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     2221 cggtaccgga gggagcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2281 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggtga
     2341 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2401 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2461 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacatcca
     2521 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2581 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2641 caacatcctg gggcacaagc tggagtacaa ccttcctgac caactgactg aagagcagat
     2701 cgcagaattt aaagaggctt tctccctatt tgacaaggac ggggatggga caataacaac
     2761 caaggagctg gggacggtga tgcgttctct ggggcagaac cccacagaag cagagctgca
     2821 ggacatgatc aatgaagtag atgccgacgg tgacggcaca atcgacttcc ctgagttcct
     2881 gacaatgatg gcaagaaaag ggagctacag ggacagtgaa gaagaaatta gagaagcgtt
     2941 cggtgtgttt gataaggatg gcaatggcta catcagtgca gcagagcttc gccacgtgat
     3001 gaacaacctt ggagagaagt taacagatga agaggttgat gaaatgatca gggaagcaga
     3061 catcgatggg gatggtcagg taaactacga agagtttgta caaatgatga cagcgaagta
     3121 gtaacttaag aattcaccgg tcagacctcc ttctccctct cttcaggtct gcagtcgacg
     3181 gtaccgcggg cccgggatcc accggatcta gataactgat cactgtggaa tcagagagac
     3241 tgcccctctc cctctccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     3301 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc
     3361 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     3421 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     3481 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     3541 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     3601 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     3661 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     3721 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
     3781 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca tggccaagtt
     3841 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     3901 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     3961 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     4021 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     4081 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     4141 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     4201 cagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg
     4261 acctgataga tccggaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga
     4321 aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt
     4381 cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt
     4441 ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt
     4501 gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg
     4561 tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttttgcagc cggtcgcgga
     4621 ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg
     4681 accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc
     4741 ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc
     4801 tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc
     4861 ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg
     4921 gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc
     4981 gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc
     5041 aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag
     5101 cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt
     5161 ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg
     5221 gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc
     5281 gagggcaaag gaataatagg gatcctgatc ataatcagcc ataccacatt tgtagaggtt
     5341 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     5401 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5461 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5521 atcaatgtat cttaggagat cccctttagt gagggttaat ttcgagcttg gcgtaatcat
     5581 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5641 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5701 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5761 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5821 ctgactcgct gcactcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5881 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5941 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6001 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6061 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6121 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6181 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6241 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6301 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6361 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6421 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6481 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6541 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6601 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6661 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6721 atgagtaaac ttggtctgac agtta
//
