LOCUS       sequence-434124-        6314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SAC1 with the TMD deleted and replaced with the Fis1tail to replace 
            the TMD and target the mitochondria instead of the ER with a Green 
            Fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6314)
  AUTHORS   Doyle CP, Rectenwald A, Timple L, Hammond GRV
  TITLE     Orthogonal Targeting of SAC1 to Mitochondria Implicates ORP2 as a 
            Major Player in PM PI4P Turnover.
  JOURNAL   Contact (Thousand Oaks). 2024 Feb 7;7:25152564241229272. doi: 
            10.1177/25152564241229272. eCollection 2024 Jan-Dec.
  PUBMED    38327560
REFERENCE   2  (bases 1 to 6314)
  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: "Contact 
            (Thousand Oaks)."; date: "2024-02-7"; volume: "7:25152564241229272. 
            doi"; pages: " 10.1177/25152564241229272. eCollection 2024 Jan-Dec"
COMMENT     Sequence Label: EGFP-1-SAC1deltaTMD-Fis1tail
FEATURES             Location/Qualifiers
     source          1..6314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3102..3223
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3139..3158)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3193..3212
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3230..3685)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3367..3388)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3579..3598
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3712..3816
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3818..4175
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3818..4009
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3841..3861)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4026..4161
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4210..5004
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4210..5004
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4264..4283)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4874..4893
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5192..5211)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5236..5283
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5612..6200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6101..6120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctatggcg acggcggcct acgagcagct gaagctgcat
     1381 atcacacctg aaaaatttta tgtggaagct tgtgatgatg gagcagatga cgtacttacc
     1441 attgaccgtg tgtccacaga ggttaccctt gcagtcaaga aagatgttcc tccttcagct
     1501 gtcacaagac caatatttgg tatactgggc acaatccatc tggtggcagg taattatctt
     1561 atagtcatta ccaaaaagat aaaagtaggt gaatttttca gtcatgtagt ctggaaagca
     1621 acagattttg atgtcctttc ttataagaag acaatgttgc acttaactga tattcagtta
     1681 caagataata aaaccttcct agcgatgcta aaccatgtct tgaatgtgga tggattttac
     1741 ttttcaacaa catatgattt gacccatact ttgcagcggc tatccaacac tagtcctgaa
     1801 ttccaagaaa tgagtctctt ggaaagggca gatcagcggt ttgtatggaa tggtcatctt
     1861 ctaagagaac tttctgcaca gccagaggtt catcggtttg cccttccagt gttacatggc
     1921 tttattacca tgcattcatg ttctattaat ggaaaatact ttgattggat tctcatctcg
     1981 aggaggagct gtttcagagc tggtgtgcgc tattatgtaa gaggaattga ttcggaaggc
     2041 catgcagcta actttgtaga aacagaacaa attgtgcact acaatgggag caaagcttcg
     2101 tttgtacaga ctcgaggatc aatacctgtt ttctggtccc aaagaccaaa cctcaagtac
     2161 aaaccactgc cacagatcag caaagtagca aatcacatgg acggtttcca aaggcatttt
     2221 gattcccaag taattattta tggaaaacaa gttataatca atctgattaa ccagaagggc
     2281 tcggagaagc cacttgagca gacatttgca acaatggtgt cttccttggg aagtggaatg
     2341 atgagataca ttgcctttga cttccataag gaatgtaaaa atatgagatg ggatcgacta
     2401 agtattttat tggatcaggt agcagaaatg caagatgaat taagttattt tctagtggac
     2461 tctgctggcc aggtggtggc aaaccaggaa ggcgtgttcc gaagcaattg catggattgt
     2521 ctagatagaa ccaatgtgat ccagagtttg ttagctcgtc gttcacttca ggcccaactt
     2581 cagagactag gagttttgca tgtgggacaa aagcttgaag aacaagatga atttgagaag
     2641 attttcaaaa atgcctgggc tgacaacgca aatgcttgtg ccaagcaata tgcgggaact
     2701 ggtgccttga agactgactt taccagaact ggaaagagaa ctcatttggg acttataatg
     2761 gatggctgga actcaatgat acgatattat aagaacaact tttccgatgg atttagacaa
     2821 gattccatag acttatttct tggaaactat tcagtggatg aattagaatc tcatagtcct
     2881 ttaagtgttc caagggactg gaaattggga ctcgtgggca tggccatcgt gggaggcatg
     2941 gccctgggtg tggcgggact ggccggactc atcggacttg ctgtgtccaa gtccaaatcc
     3001 tgatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3061 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3121 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3181 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc
     3241 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3301 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3361 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3421 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3481 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3541 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3601 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3661 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3721 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3781 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     3841 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3901 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3961 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4021 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4081 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4141 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     4201 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4261 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4321 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4381 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4441 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4501 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4561 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4621 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4681 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     4741 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4801 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4861 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4921 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4981 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5041 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5101 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5161 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     5221 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     5281 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     5341 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     5401 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     5461 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5521 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5581 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5641 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5701 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5761 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5821 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5881 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5941 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6001 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6061 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6121 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6181 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6241 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6301 ttaccgccat gcat
//
