LOCUS       sequence-114840-        6931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This is a 2A peptide construct that produces two different proteins 
            from a single open reading frame, a nuclear targeted Cre 
            recombinase, and a membrane targeted GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6931)
  TITLE     Cre Shine
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6931)
  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            "Unpublished"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: Cre Shine
FEATURES             Location/Qualifiers
     source          1..6931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        10..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     638..657
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        638..656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             736..1764
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             1765..1818
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1888..1890,1891..1893,1894..2607)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1888..1890,1891..1893,1894..2604)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2194..2213)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2541..2562
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2684..2701)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2690..2914
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3046..3634)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3126..3145)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3963..4010
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4035..4054
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4242..5030)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDQEHQGL
                     APAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIALA
                     TRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4353..4372)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4957..4976
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5065..5422)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5079..5214
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5231..5422
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5379..5399
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5482..6273,6274..6342))
                     /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     6105..6124
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6343..6447)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6474..6929
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6561..6580)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6771..6792
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      601 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
      661 cccaagctgg ctagcgttta aacttaagct tggtaccgag ctcggatcca ctagtccagt
      721 gtggtggaat tcaccatgtc caatttactg accgtacacc aaaatttgcc tgcattaccg
      781 gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg acatgttcag ggatcgccag
      841 gcgttttctg agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg
      901 tgcaagttga ataaccggaa atggtttccc gcagaacctg aagatgttcg cgattatctt
      961 ctatatcttc aggcgcgcgg tctggcagta aaaactatcc agcaacattt gggccagcta
     1021 aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc tgtttcactg
     1081 gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta
     1141 gcgttcgaac gcactgattt cgaccaggtt cgttcactca tggaaaatag cgatcgctgc
     1201 caggatatac gtaatctggc atttctgggg attgcttata acaccctgtt acgtatagcc
     1261 gaaattgcca ggatcagggt taaagatatc tcacgtactg acggtgggag aatgttaatc
     1321 catattggca gaacgaaaac gctggttagc accgcaggtg tagagaaggc acttagcctg
     1381 ggggtaacta aactggtcga gcgatggatt tccgtctctg gtgtagctga tgatccgaat
     1441 aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc caccagccag
     1501 ctatcaactc gcgccctgga agggattttt gaagcaactc atcgattgat ttacggcgct
     1561 aaggatgact ctggtcagag atacctggcc tggtctggac acagtgcccg tgtcggagcc
     1621 gcgcgagata tggcccgcgc tggagtttca ataccggaga tcatgcaagc tggtggctgg
     1681 accaatgtaa atattgtcat gaactatatc cgtaacctgg atagtgaaac aggggcaatg
     1741 gtgcgcctgc tggaagatgg cgatgagggc agaggaagtc ttctaacatg cggtgacgtg
     1801 gaggagaatc ccgggcctat gctgtgctgc ataagaagaa ccaaaccggt ggagaaaaat
     1861 gaggatgcag accagaagat tctcgagatg gtgagcaagg gcgaggagct gttcaccggg
     1921 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1981 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2041 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2101 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2161 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2221 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2281 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     2341 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2401 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2461 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     2521 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2581 ctcggcatgg acgagctgta caagtaactc gaggaattct gcagatatcc agcacagtgg
     2641 cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     2701 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2761 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2821 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2881 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagtggc
     2941 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3001 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3061 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3121 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3181 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3241 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3301 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3361 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3421 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3481 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3541 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3601 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3661 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3721 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3781 atatgagtaa cctgaggcta tggcagggcc tgccgccccg acgttggctg cgagccctgg
     3841 gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa gaaacgcggg cgtattggcc
     3901 ccaatggggt ctcggtgggg tatcgacaga gtgccagccc tgggaccgaa ccccgcgttt
     3961 atgaacaaac gacccaacac cgtgcgtttt attctgtctt tttattgccg tcatagcgcg
     4021 ggttccttcc ggtattgtct ccttccgtgt ttcagttagc ctccccctag ggtgggcgaa
     4081 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat
     4141 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt
     4201 gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga
     4261 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     4321 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     4381 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     4441 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg
     4501 ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttga
     4561 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     4621 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     4681 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     4741 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     4801 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcttg cagttcattc
     4861 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     4921 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     4981 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     5041 cctcatcctg tctcttgatc gatctttgca aaagcctagg cctccaaaaa agcctcctca
     5101 ctacttctgg aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat
     5161 tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg
     5221 agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc
     5281 tgctggggag cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg
     5341 catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca
     5401 cagctggttc tttccgcctc aggactcttc ctttttcaat aaatcaatct aaagtatata
     5461 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5521 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5581 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5641 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5701 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5761 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5821 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5881 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5941 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6001 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6061 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6121 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6181 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6241 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6301 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6361 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6421 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
     6481 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     6541 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     6601 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     6661 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     6721 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     6781 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     6841 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     6901 ttttaacaaa atattaacgc ttacaattta c
//
