LOCUS       sequence-474717-        8815 bp    DNA     circular SYN 05-JUN-2025
DEFINITION  Express LgBiT-CALM1/SmBiT-ADCY1.1 Fusion Protein in Mammalian Cells 
            under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8815)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 8815)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: LgBiT-CALM1/SmBiT-ADCY1.1 BiBiT Vector
FEATURES             Location/Qualifiers
     source          1..8815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    3423..3544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3434..3453)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3659..3692)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3702..3749
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3781..4575
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3835..3854)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4445..4464
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4733..5321)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4813..4832)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5447..5495
                     /note="synthetic polyadenylation signal"
     CDS             complement(5519..5917)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5914..5923
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5948..6305)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5962..6097
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6114..6305
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6262..6282
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    6384..6465
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6668..7117)
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPT
                     EAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAA
                     ELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     promoter        complement(7687..7705)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7688..7705)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        7825..8128
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8129..8332
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8283..8303
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          8468..8600
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     8583..8602
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     8644..8663
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8644..8662
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gcccgggggc gccgagcggg cggccgggac aagccgccgg cgcgggctcc gggcgtgcga
       61 cgaggagttc gcttgcccag agctggaggc gctgttccgc ggctacacgc tgcggctgga
      121 gcaggcggcc acgctgaagg cgctggccgt tctcagcctg ctggcgggcg cgctggcgct
      181 ggccgagctg ctgggcgcgc cggggcccgc gcccggcctg gccaagggct cacacccggt
      241 gcactgcgtc ctcttcctgg cgctgctcgt ggtaaccaac gtccggtccc tgcaggtgcc
      301 ccagctgcag caggtcggcc agctggcgct gctcttcagc ctcaccttcg cgctgctctg
      361 ctgtcctttc gcgctgggcg gccccgcccg gggttccgcc ggggccgctg gggggccagc
      421 gaccgccgaa caaggggttt ggcagctcct tttggtcacc ttcgtgtcct atgccttgct
      481 gcccgtgcgc agcctgctgg ccataggctt tgggctcgtg gtggctgcgt cgcacttgct
      541 ggtcacagcc accttggtcc ccgccaagcg cccacgtctc tggaggacgc tcggtgccaa
      601 tgccttgctc ttcgtcggtg tgaacatgta tggggtcttt gtgcggattc tgactgagcg
      661 ttcacagagg aaggcgttcc tgcaggcccg gagctgcatt gaggaccgac tgaggctgga
      721 ggatgagaac gagaagcagg agcggctcct catgagcctc ctgccccgga acgttgccat
      781 ggagatgaag gaggacttcc tgaagccccc tgagaggatt ttccacaaga tttacatcca
      841 gaggcacgac aatgtgagca tcctgtttgc tgacatcgtg ggtttcacgg gcttggcatc
      901 ccagtgcaca gcccaggagc tggtgaaact cctcaatgag ctcttcggca agttcgatga
      961 attagccacg gagaaccact gtcgccgcat caagattctc ggggactgct actactgcgt
     1021 gtcgggcctc acccagccca agactgacca tgcccactgc tgtgtggaga tgggactcga
     1081 catgattgat accatcacat ctgtggctga agccaccgag gtggatctga acatgcgtgt
     1141 gggtctgcac acgggcaggg tcctctgtgg tgtcctgggc ttgcgcaagt ggcagtacga
     1201 cgtgtggtcc aatgatgtga ccttggccaa tgtcatggaa gccgctggcc tgccagggaa
     1261 ggttcatatc acaaagacga ccctagcgtg cttgaatggg gactacgagg tagaaccggg
     1321 ttacggacat gagaggaaca gtttcttgaa aactcataac atcgaaacct tttttattgt
     1381 gccatcccat cgccgaaaga tatttccagg cctgattctc tcagatataa aaccggccaa
     1441 aaggatgaag ttcaagactg tctgctacct gctggtgcag ctcatgcact gccggaaaat
     1501 gttcaaggcc gagatcccct tctccaatgt catgacctgc gaggacgatg acaagcggag
     1561 ggcattaaga acagcctcgg aaaaactcag aaaccgctca tctttttcta ccaacgttgt
     1621 ctacaccacc ccgggcactc gcgtcaacag gtacatcagc cgcctcttag aagcccgcca
     1681 gacagagctg gagatggcag acctgaactt ctttaccctg aagtacaaac atgtcgaacg
     1741 ggagcaaaag taccaccagc ttcaggacga gtatttcacc agcgccgttg tcctcaccct
     1801 catcctggct gccttatttg gccttgtcta ccttctaata ttcccacaga gtgtggtcgt
     1861 cctgctcctg ctagtattct gcatctgctt cctggtggcc tgtgtcctgt acctgcacat
     1921 cacccgggtc cagtgttttc cagggtgcct gacgattcag attcgcactg tcctgtgtat
     1981 tttcatagtg gtcttaatct actcagtagc ccaaggttgt gtggtgggct gcctgccttg
     2041 ggcctggagc tccaagccca acagttccct ggtggtcctt tcgtctgggg gccagcgcac
     2101 agccctgccc accctgccct gcgagtctac acaccatgcc ctgctctgct gcctggtggg
     2161 caccctcccg ctagccatat ttttccgggt gtcctccttg ccaaaaatga tcctgctctc
     2221 cgggctcacc acgtcctaca tcctcgttct ggagctcagc ggatacacca ggactggggg
     2281 tggtgccgtc tccgggcgca gctacgagcc gattgtggcc atcctgctct tctcctgtgc
     2341 gctggccctg catgccaggc aggtggacat caggctgagg ctggactacc tctgggccgc
     2401 acaggcagag gaggagcgag aggacatgga gaaggtgaag ctggacaaca ggcgcatcct
     2461 cttcaacctc ctgccggccc acgtcgccca gcacttcctc atgtccaacc ctcggaacat
     2521 ggacctctac taccagtcct actcccaggt gggcgtcatg tttgcctcca tccccaactt
     2581 caatgacttc tacatcgagc tggacggcaa caacatgggg gtggagtgtc tgcggcttct
     2641 caacgagatc atcgccgact ttgacgagct catggaaaaa gacttttaca aggacataga
     2701 gaagatcaag accatcggga gcacctacat ggccgctgtg gggctagcgc ccacctcggg
     2761 gaccaaggct aagaagtcca tctcctccca cctgagcacg ctggcggact ttgccattga
     2821 gatgtttgac gttctggatg aaatcaacta ccagtcttac aacgactttg tcctccgagt
     2881 tggcatcaat gttggccctg tggtggctgg agtgattggc gctcgcaggc cccagtacga
     2941 catctgggga aacacagtca acgtggccag tcggatggat agcacagggg tccagggcag
     3001 aatccaggtg actgaggaag tccaccggct gctgagaagg tgcccctacc actttgtgtg
     3061 ccgaggcaaa gtcagtgtca agggcaaagg cgagatgttg acatactttc tagaaggcag
     3121 gactgatgga aacggctccc aaatcaggtc cctgggcttg gatcggaaaa tgtgtccatt
     3181 tgggagagct ggccttcagg gcagacgtcc ccccgtgtgc cccatgcctg gcgtctcagt
     3241 cagggctggg ctccctccac actccccagg ccagtacctg ccctctgcag cagctgggaa
     3301 ggaggcttga gtttaaacga attcgggctc ggtacccggg gatcctctag agtcgacctg
     3361 caggcatgca agctgatttg gctgctgcca ccgctgagca aggccgcttc gagcagacat
     3421 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     3481 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     3541 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
     3601 ttttttaagc aagtaaaacc tctacaaatg tggtaaaatc ttaattaaga attctaatat
     3661 aacttcgtat agcatacatt atacgaagtt atggatccac tgttgacaat taatcatcgg
     3721 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaacccag gaggcagatc
     3781 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3841 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3901 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3961 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4021 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4081 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4141 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4201 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4261 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4321 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4381 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4441 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4501 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4561 gacgagttct tctgattcga aatgaccgac caagcgacgc ccaaccggta tcagctcact
     4621 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4681 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4741 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4801 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4861 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4921 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4981 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5041 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5101 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5161 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5221 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5281 tttgcaagca gcagattacg cgcagaaaaa aaggatttca agaagatcct ttgatctttt
     5341 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5401 tatcaaaaag gatcttcacc tagatccttt tatagtccgg aatcgtcaca caaaaaacca
     5461 acacacagat gtaatgaaaa taaagatatt ttattgcggc catcgtgatg gctagagttt
     5521 agccctccca cacataacca gagggcagca attcacgaat cccaactgcc gtcggctgtc
     5581 catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc acctgtcggc
     5641 accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg atacaagtca
     5701 ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca caaggtcccc
     5761 cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag agagctgcgc
     5821 tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt gctctttcaa
     5881 tgagggtgga ttcttcttga gacaaaggct tggccatggt ggcgctagtg tcagaagaat
     5941 cgagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     6001 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     6061 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     6121 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     6181 actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     6241 gggagcctgg ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc
     6301 ctcagaaggt acctaaccaa gttcctcttt cagaggttat ttcaggccat ggtgctgcgc
     6361 aagacgcgtt aattaagacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
     6421 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     6481 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     6541 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
     6601 tgacttcgta cgagagccta ggtgtttcta gaggatcccc gggtaccgag cccgaattcg
     6661 tttaaactca ttttgcagtc atcatctgta cgaattcttc atagttgact tgtccgtctc
     6721 catcaatatc tgcttctctg atcatttcat ctacttcttc atctgttagt ttttctccta
     6781 agtttgtcat gacgtgacgt agttctgctg cactgatata accattgcca tccttgtcaa
     6841 agactcggaa tgcctcacgg atttcttctt cactatctgt atctttcatt tttctagcca
     6901 tcatagtcaa aaattcgggg aagtcaatgg tgccattacc atcagcatcc acttcattga
     6961 tcatatcctg caattcagct tctgttgggt tctgacccag tgacctcatg acagttccaa
     7021 gttcctttgt tgtgatggtg ccatcgccat ctttatcaaa tagggagaag gcttccttga
     7081 attcagcaat ctgttcttcg gtcagctgat cagccatggc gatcgcaccg ctcgagcctc
     7141 cacctccgct cccgccacca ccggagctcc cactgttgat ggttactcgg aacagcatgg
     7201 agccgtcggg ggtgatcagg cgctcgtcga taattttgtt gccgttccac agggtccctg
     7261 ttacagtgat ctttttgccg tcgaacacgg cgatgccttc atacggccgt ccgaaatagt
     7321 tcagcatgtt cggcgtaacc ccgtcgatta ccagtgtgcc atagggcagg atcaccttaa
     7381 agtgatgatc atccacaggg tacaccacct taaacacctc ttcgatctgg gccatttggt
     7441 cggcgctcag accttcatac gggatgatga catggatgtc gatcttcagg gcattttcac
     7501 cgctccggac aatcctttgg atcggagtta cggacacggc gagattctgc agcaaactgg
     7561 acacacctcc ctgttcaagg acttggtcca ggttgtaggc ggctgtctgt tcccagtccc
     7621 caacgaaatc ttcgagtgtg aagaccatgg tgagcgctag ctgcaaaaag aacaagtagc
     7681 ttgtattcta tagtgtcacc taaatgggcg atctgacggt tcactaaacc agctctgctt
     7741 atataggcct ccctggcgcg ccactagtta ttaatagtaa tcaattacgg ggtcattagt
     7801 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     7861 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     7921 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     7981 agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg
     8041 gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat
     8101 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg
     8161 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     8221 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt
     8281 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     8341 gaaccgtcag atcactagaa gctttattgc ggtagtttat cacagttaaa ttgctaacgc
     8401 agtcagtgct tctgacacaa cagtctcgaa cttaagctgc agaagttggt cgtgaggcac
     8461 tgggcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt
     8521 gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca
     8581 ctttgccttt ctctccacag gtgtccactc ccagttcaat tacagctctt aaggctagag
     8641 tattaatacg actcactata gggctagcgc tcaccatggt gaccggctac cggctgttcg
     8701 aggagattct cgggagctcc ggtggtggcg ggagcggagg tggaggctcg agcggtgcga
     8761 tcgccatggc gggggcgccg cgcggcggag gcggcggcgg aggcggcgcg ggcga
//
