LOCUS       sequence-466031-        5857 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of large NanoBit fragment (LgBit) .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5857)
  AUTHORS   Jang W, Senarath K, Feinberg G, Lu S, Lambert NA
  TITLE     Visualization of endogenous G proteins on endosomes and other 
            organelles.
  JOURNAL   Elife. 2024 Nov 8;13:RP97033. doi: 10.7554/eLife.97033.
  PUBMED    39514269
REFERENCE   2  (bases 1 to 5857)
  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: "Elife."; 
            date: "2024-11-8"; volume: "13:RP97033. doi"; pages: " 
            10.7554/eLife.97033"
COMMENT     Sequence Label: CMV-LgBit
FEATURES             Location/Qualifiers
     source          1..5857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1335..2129)
                     /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(1446..1465)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2056..2075
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2196..2525)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2210..2345
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2264..2283)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2510..2530
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2539..2967)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2649..2670)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2861..2880
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3013..3237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3226..3243
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     regulatory      3754..3763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3809..3828)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3810..3828)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3873..4076)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3902..3922)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4077..4456
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4628..4647
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4722..4826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4827..4895,4896..5687)
                     /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     complement(5045..5064)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     1321 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1381 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1441 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1501 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     1561 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     1621 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     1681 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     1741 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     1801 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     1861 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     1921 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     1981 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     2041 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2101 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2161 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     2221 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     2281 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     2341 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     2401 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     2461 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     2521 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2581 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2641 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2701 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2761 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2821 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2881 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2941 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     3001 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     3061 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     3121 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     3181 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     3241 aggctgatca gcgggtttaa acgggccctc tagactcgag ttagttgatg gttactcgga
     3301 acagcatgga gccgtcgggg gtgatcaggc gctcgtcgat aattttgttg ccgttccaca
     3361 gggtccctgt tacagtgatc tttttgccgt cgaacacggc gatgccttca tacggccgtc
     3421 cgaaatagtt cagcatgttc ggcgtaaccc cgtcgattac cagtgtgcca tagggcagga
     3481 tcaccttaaa gtgatgatca tccacagggt acaccacctt aaacacctct tcgatctggg
     3541 ccatttggtc ggcgctcaga ccttcatacg ggatgatgac atggatgtcg atcttcaggg
     3601 cattttcacc gctccggaca atcctttgga tcggagttac ggacacggcg agattctgca
     3661 gcaaactgga cacacctccc tgttcaagga cttggtccag gttgtaggcg gctgtctgtt
     3721 cccagtcccc aacgaaatct tcgagtgtga agaccatggt ggctctagat tgccaagctt
     3781 aagtttaaac gctagccagc ttgggtctcc ctatagtgag tcgtattaat ttcgataagc
     3841 cagtaagcag tgggttctct agttagccag agagctctgc ttatatagac ctcccaccgt
     3901 acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc
     3961 cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat
     4021 ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg
     4081 gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg
     4141 tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt
     4201 ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat
     4261 tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc
     4321 aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg
     4381 cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta
     4441 gtcaataatc aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc
     4501 ctaaccctaa gcagattctt catgcaattg tcggtcaagc cttgccttgt tgtagcttaa
     4561 attttgctcg cgcactactc agcgacctcc aacacacaag cagggagcag atactggctt
     4621 aactatgcgg catcagagca gattgtactg agagtgcacc ataggggatc gggagatctc
     4681 ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4741 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4801 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     4861 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     4921 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     4981 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5041 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5101 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5161 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5221 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5281 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5341 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5401 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5461 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5521 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5581 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5641 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5701 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5761 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5821 gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
