LOCUS       sequence-423484-        7672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses equimolar Nucleus- and Mitochondria- targeted split-FAST 
            fragments for Short-range NU-MT Contact sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7672)
  AUTHORS   Cali T
  TITLE     SPLICSFAST-P2A
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7672)
  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: 
            "Unpublished"
COMMENT     Sequence Label: SPLICSFAST-NU-MT-P2A
FEATURES             Location/Qualifiers
     source          1..7672
                     /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             2641..2697
                     /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             2707..2736
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      2743..2752
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3263..3280)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3269..3493
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3539..3967
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3626..3645)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3836..3857
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3976..3996)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3981..4310
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4161..4296
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4223..4242
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4377..5171
                     /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(4431..4450)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5041..5060
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5345..5466
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5382..5401)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5436..5455
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5515..5531)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5515..5531)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5528..5550)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5539..5555
                     /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(5563..5569,5570..5587,5588..5593))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5608..5629
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5746..5763)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5917..6505)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5997..6016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6676..7467,7468..7536))
                     /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     7299..7318
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7537..7641)
                     /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 ccatgggcct gttcagagtg tgcctggtgg tggtgaccgc catcatcaac
      961 caccccctgc tgttcccccg cgagaacgcc accgtgcctg agaatgagga agagatcatc
     1021 agaaagatgc aagctcacca ggagaagctg cagctcgaac aactccggct ggaagaagaa
     1081 gtggctcggc tggccgctga gaaggaagcc ctggagcagg tggccgagga aggcagacag
     1141 cagaacgaga caagagtggc ctgggacctg tggtccaccc tgtgcatgat cctgttcctg
     1201 atgattgagg tgtggcggca ggaccaccag gagggaccaa gccccgagtg cctgggcggc
     1261 gaggaagacg agctgcctgg cctgggcggc gctcctctgc agggcttgac actgccaaac
     1321 aaggccaccc tgggccactt ttacgagcgt tgcatcagag gcgccaccgc cgatgccgcc
     1381 agaaccagag agttcctgga gggcttcgtg gatgatctgc tggaagccct gagaagcctg
     1441 tgtaacagag ataccgatat ggaagtcgag gacttcatcg gcgtggacag catgtacgaa
     1501 aactggcagg tggacagacc cctgctttgc cacctgttcg ttccattcac ccctcctgag
     1561 ccctaccggt tccaccctga actgtggtgc agcggccggt ccgtgcccct ggaccggcag
     1621 ggatatggcc agatcaaggt cgtcagagcc gacggcgaca ccctgtcttg tatctgcggc
     1681 aaaacaaagc tgggagagga catgctgtgc ctgctgcacg gcagaaatag catggcccct
     1741 ccatgtggcg acatggaaaa tctgctgtgc gccacagatt ctctgtacct ggacaccatg
     1801 caggtgatga aatggttcca aaccgccctg acccgggcct ggaagggcat cgcccataag
     1861 tacgagtttg acctcgcttt tggtcagctg gatagccctg gcagcctgaa gatcaaattc
     1921 cggtcaggca agttcatgcc tttcaacctg attcctgtga tccagtgcga cgacagcgat
     1981 ctgtatttcg tgtcccacct gcctagagag cctagcgagg gaacacctgc cagcagcacc
     2041 gactggctgc tgagcttcgc tgtgtacgag agacactttc tgcgaaccac cctgaaagct
     2101 ctgcctgaag gcgcctgcca cctgagctgt ctgcagatcg cctcgttcct gctgagcaag
     2161 cagagcagac tgacaggacc atctggcctg tcctcctacc acctgaagac cgccctgctg
     2221 cacctgctgc tgctgagaca ggccgctgac tggaaagccg gccagctgga cgccagactg
     2281 cacgagctgc tgtgcttcct ggaaaagagc ctgctgcaaa agaagctgca ccacttcttc
     2341 atcggcaata gaaaggtgcc cgaggctatg ggtctgccag aagccgtgct gagagccgag
     2401 cccctcaacc tttttcggcc tttcgtgctg cagagatctt tatacaggaa gactttggac
     2461 agcttttacg agatgctcaa gaacgcccct gccctgatca gcgagtacag cctgcatgtg
     2521 ccttctgatc agcctacacc taagagcagc ggtggagatg gcggcagcgg aggtggctct
     2581 atgggcgaca gctactgggt attcgttaag cggctgaagg aactggcgtg tgggagcggc
     2641 gccacaaact tcagcctcct gaaacaggcc ggcgatgtgg aagagaaccc cggccctaga
     2701 tccatggaac agaagctgat ctctgaggag gacctggaat tcgccgccat ggtgggcaga
     2761 aactctgcca tcgccgccgg agtgtgtggc gctctgttca tcggatactg catctacttc
     2821 gacagaaagc gtagatctga tcctaacagc ggaggcggtg gatctggtgg tggcggatca
     2881 atggaacacg tggccttcgg ctctgaggat atcgaaaaca ccctggccaa gatggacgat
     2941 ggccagcttg atggcctggc ctttggcgcc atccagctgg atggcgacgg aaatatcctg
     3001 cagtacaacg ccgcagaagg cgacatcacc ggccgggacc ccaagcaggt gatcggaaaa
     3061 aacttcttta aggacgtggc accaggcacc gacagccctg agttctacgg caagttcaag
     3121 gagggagtcg ccagcggcaa cctgaacaca atgttcgagt ggatgatccc cacatcccgg
     3181 ggccctacca aggtgaaggt gcacatgaaa aaggccctga gctgactcga gtctagaggg
     3241 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3301 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3361 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3421 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3481 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     3541 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3601 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3661 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3721 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3781 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3841 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3901 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3961 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     4021 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     4081 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     4141 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4201 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     4261 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     4321 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     4381 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4441 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4501 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     4561 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4621 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4681 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4741 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4801 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4861 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     4921 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4981 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     5041 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     5101 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     5161 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     5221 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     5281 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5341 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5401 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5461 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5521 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5581 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5641 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5701 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5761 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5821 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5881 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5941 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6001 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6061 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6121 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6181 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6241 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6301 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6361 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6421 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6481 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6541 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6601 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6661 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6721 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6781 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6841 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6901 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6961 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     7021 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7081 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7141 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7201 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7261 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7321 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7381 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7441 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7501 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7561 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7621 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
