LOCUS       sequence-423481-        6931 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses equimolar Lipid Droplet- and Lysosome- targeted split-FAST
            fragments for Short-range LD-LY Contact sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6931)
  AUTHORS   Cali T
  TITLE     SPLICSFAST-P2A
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6931)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: SPLICSFAST-LDh-LY-P2A
FEATURES             Location/Qualifiers
     source          1..6931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1165..1221
                     /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     complement(2522..2539)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2528..2752
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2798..3226
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2885..2904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3095..3116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3235..3255)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3240..3569
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3420..3555
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3482..3501
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3636..4430
                     /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(3690..3709)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4300..4319
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4604..4725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4641..4660)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4695..4714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4774..4790)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4774..4790)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4787..4809)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4798..4814
                     /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(4822..4828,4829..4846,4847..4852))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4867..4888
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5005..5022)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5176..5764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5256..5275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5935..6726,6727..6795))
                     /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     6558..6577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6796..6900)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatgg gcgacagcta ctgggtcttt gtgaagagag agttcggcgg agatggcggc
      961 agcggcggag gagatatttc taagctgatg gacgtgctcg tgcccctgct gcaactgctg
     1021 gttctgctgc tgaccctgcc actgcacctg ctcgccctgc tgggctgctg gcagccactg
     1081 ttcgaggcga tcggcaaaat cttcagcaac atcagaatct ctacccagaa ggaaatcctg
     1141 aaagagctgg catgcggcag cggggccacc aatttcagcc tgctgaagca agccggcgac
     1201 gtggaagaaa accccggccc aagaagcacc atggccgctg gcggaagaat ggaagatgga
     1261 agcctggata tcacccagag catcgaggac gatcctctgc tggacgccca gctgctgcct
     1321 caccatagcc tgcaggccca cttccggcct agattccacc ccctgcctac agtgattatc
     1381 gtgaacctgc tgtggttcat ccacctggtg ttcgtggtgc tggccttcct gacaggcgtg
     1441 ttgtgcagct accctaatcc taacgaggac aagtgccctg gcaactacac aaaccctctg
     1501 aaagtgcaga ccgtgatcat cctgggaaag gtgatccttt ggatcctgca cctgctcctg
     1561 gaatgctaca tccagtacca ccactccaaa attcggaaca gaggctacaa cctgatctac
     1621 cggagcacaa gacatctgaa gcggctggcc ctgatgatcc aatctagcgg caataccgtg
     1681 ctgctgctta tcctttgtat gcagcacagc ttcccggagc ctggaagact gtacctggac
     1741 ctgatcctgg ccatcctggc cctggagctg atctgcagcc tgatttgtct gctgatctac
     1801 accgtgaaga tccgcagatt caacaaggcc aagcctgagc ccgacatcct ggaagaggaa
     1861 aagatctatg cctaccccag taacatcaca tctgaaaccg gcttcagaac catcagctct
     1921 ctcgaagaga tcgtcgagaa gcagggcgac accatcgagt acctgaagag gcacaacgcc
     1981 ctgctcagca agcggctgct ggccctgacc agcagcgacc tgggctgtca gcctagcaga
     2041 acagacgtgg gcggaggtgg ctctgagggc ggtggatctg gcggccctgg ctccggcggc
     2101 gagggaagcg ctggtggcgg atcagccgga ggcgggagca tggaacacgt ggcctttggc
     2161 tccgaggata tcgagaacac cctggctaag atggacgacg gacagctgga cggcctggct
     2221 tttggcgcca tccagctgga cggggatgga aatatcctgc agtataacgc cgccgagggc
     2281 gacatcacag gccgggaccc caagcaggtg atcggcaaga acttcttcaa ggacgtggcc
     2341 cctggtacag atagccctga gttttacggc aagttcaagg aaggcgtggc ttctggaaat
     2401 ctgaacacca tgttcgagtg gatgatccct acctccagag gccccaccaa ggtgaaagtg
     2461 cacatgaaaa aggccctgag ctgactcgag tctagagggc ccgtttaaac ccgctgatca
     2521 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2581 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2641 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2701 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2761 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     2821 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2881 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2941 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3001 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3061 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3121 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3181 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3241 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3301 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3361 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3421 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3481 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     3541 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     3601 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3661 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3721 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3781 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     3841 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3901 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3961 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4021 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4081 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4141 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     4201 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4261 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4321 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4381 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4441 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4501 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4561 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     4621 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4681 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     4741 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4801 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4861 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4921 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4981 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5041 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5101 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5161 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5221 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5281 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5341 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5401 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5461 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5521 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5581 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5641 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5701 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5761 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5821 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5881 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5941 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6001 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6061 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6121 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6181 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6241 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6301 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6361 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6421 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6481 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6541 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6601 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6661 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6721 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6781 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6841 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6901 cacatttccc cgaaaagtgc cacctgacgt c
//
