LOCUS       sequence-180617-        7662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of IRFP720 using PGK promoter in a 3rd 
            generation lentiviral backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7662)
  TITLE     Torok-Storb lab lentiviral fluorescent protein
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7662)
  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            "Unpublished"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: pRRLsinPGK_IRFP720_WPRE
FEATURES             Location/Qualifiers
     source          1..7662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..52)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(206..794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(286..305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(965..1756,1757..1825))
                     /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     1588..1607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1826..1930)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1956..2411)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2093..2114)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2305..2324
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2538..2560
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2552..2569
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2553..2569
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2579..2598
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2579..2597
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2618..2753
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2793..2914
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2804..2823)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2858..2877
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             2986..3219
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3306..3894
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3412..3423
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3821..3841
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3958..4908)
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 720 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP720
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     regulatory      4905..4914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4925..5435)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4988..5006)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    5481..5598
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(5865..5909)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6094..6327
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6820..6945
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6992..7172
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(7173..7399)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(7427..7447)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7427..7445)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7466..7482)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7466..7482)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7479..7501)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7490..7506
                     /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(7514..7520,7521..7538,7539..7544))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
       61 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      121 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      181 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      241 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      301 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      361 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      421 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      481 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      541 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      601 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      661 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      721 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      781 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      841 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
      901 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
      961 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1021 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1081 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1141 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1201 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1261 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1321 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1381 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1441 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1501 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1561 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1621 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1681 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     1741 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     1801 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     1861 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1921 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     1981 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2041 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2101 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2161 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     2221 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2281 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     2341 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     2401 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     2461 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     2521 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
     2581 atacgactca ctatagggcg aattgggtac gtccctaggc ctccaaaaaa gcctcctcac
     2641 tacttctgga atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt
     2701 agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga
     2761 gttaggggcg ggatagctag agccagacat gataagatac attgatgagt ttggacaaac
     2821 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     2881 atttgtaacc attataagct gcaataaaca agttcctctc actctctgat attcatttct
     2941 ttgcaagtta taaatactga ataataagat gacatgaact actactgcta gagattttcc
     3001 acactgacta aaagggtctg agggatctct agttaccaga gtcacacaac agacgggcac
     3061 acactacttg aagcactcaa ggcaagcttt attgaggctt aagcagtggg ttccctagtt
     3121 agccagagag ctcccaggct cagatctggt ctaaccagag agacccagta caagcaaaaa
     3181 gcagatcttg tcttcgttgg gagtgaatta gcccttccag tccccccttt tcttttaaaa
     3241 agtggctaag atctacagct gccttgtaag tcattggtct taaaggtacc gagctcgaat
     3301 tccaggcggg gaggcggccc aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg
     3361 cggaagaggc cgcagagccg gcagcaggcc gcgggaagga aggtccgctg gattgagggc
     3421 cgaagggacg tagcagaagg acgtcccgcg cagaatccag gtggcaacac aggcgagcag
     3481 ccatggaaag gacgtcagct tccccgacaa caccacggaa ttgtcagtgc ccaacagccg
     3541 agcccctgtc cagcagcggg caaggcaggc ggcgatgagt tccgccgtgg caatagggag
     3601 ggggaaagcg aaagtcccgg aaaggagctg acaggtggtg gcaatgcccc aaccagtggg
     3661 ggttgcgtca gcaaacacag tgcacaccac gccacgttgc ctgacaacgg gccacaactc
     3721 ctcataaaga gacagcaacc aggatttata caaggaggag aaaatgaaag ccatacggga
     3781 agcaatagca tgatacaaag gcattaaagc agcgtatcca catagcgtaa aaggagcaac
     3841 atagttaaga ataccagtca atctttcaca aattttgtaa tccagaggtt gattgtcgac
     3901 tgcagttcga acccgggcat atgtctagaa ccggtggatc tctagagtcg cggccgctca
     3961 ctcttccatc acgccgatct gccaggccag aacctgggcg actagctcgc aggcttggcg
     4021 gccatcgaga tcgacgtagt acggcgttcg gtgatggcaa acgatcaatc cccacagtcg
     4081 ctcgccgcgc aaaatcgaga tcgacatcgt gccgtgcatg cctatgttgc gcatgaactc
     4141 cagatggttg ggcgagacgc tgcgcaggat ggcgaagcta agatcaatcg gccgcccggt
     4201 gaccggattg aggtctgggg tgaccggcac cggccgataa ttgatatcgg gaatgatccg
     4261 taccgggttg atggtataga gccgacgggc ctgcgccggg atgaatgagg caggatagtg
     4321 caggcctagt tttgactcga cctcggcgca ccgatcctct gcgatcacgg acccgctgaa
     4381 gtcggaggcg aagcgataga tcatcacccg atcgaagccg gtaatcttcc gcacctcttg
     4441 cgccgcggcg gcgcaggcgc tttccaaggt ttcggcggcc tgcaggcggc ggatggcgct
     4501 gttggtgcgg cggaagaacg cctgcggctc ggcgacgtcc cgctggggag gctcgagctc
     4561 gaggaagatg agctgatcat ggcgatgcca ggagccgatg aagcctgcgt cctttcgcat
     4621 cgtgaagccg acagtgatcg gtgctccgac ggccgccccg ggctcggcca aggcgatcgt
     4681 cagacggttg tgcgtctccg agtcgaagac atcggccgca gagcggccga tcagggcgcc
     4741 gatcgccagt ccggtgagtt cgggaaggtt gtcgctgccg gcaacgatcg tcatgtcggc
     4801 ggcgagggcg agcagcagtc catgcggttg gatggcaccg gggatatgga tcggctcatc
     4861 gtcgcaggtc aagaggtcag gctgcctggc gacggatcct tccgccatgg tggcgaccgg
     4921 tcccctgggg agagaggtcg gtgattcggt caacgaggga gccgactgcc gacgtgcgct
     4981 ccggaggctt gcagaatgcg gaacaccgcg cgggcaggaa cagggcccac actaccgccc
     5041 cacaccccgc ctcccgcacc gccccttccc ggccgctgct ctcggcgcgc cctgctgagc
     5101 agccgctatt ggccacagcc catcgcggtc ggcgcgctgc cattgctccc tggcgctgtc
     5161 cgtctgcgag ggtactagtg agacgtgcgg cttccgtttg tcacgtccgg cacgccgcga
     5221 accgcaagga accttcccga cttaggggcg gagcaggaag cgtcgccggg gggcccacaa
     5281 gggtagcggc gaagatccgg gtgacgctgc gaacggacgt gaagaatgtg cgagacccag
     5341 ggtcggcgcc gctgcgtttc ccggaaccac gcccagagca gccgcgtccc tgcgcaaacc
     5401 cagggctgcc ttggaaaagg cgcaacccca accccgtgga attcgatatc aagcttctcg
     5461 aggctagtct cgtgatcgat aaaattttga atttttgtaa tttgtttttg taattcttta
     5521 gtttgtatgt ctgttgctat tatgtctact attctttccc ctgcactgta ccccccaatc
     5581 cccccttttc ttttaaaagt taaccgatac cgtcgagatc cgttcactaa tcgaatggat
     5641 ctgtctctgt ctctctctcc accttcttct tctattcctt cgggcctgtc gggtcccctc
     5701 ggggttggga ggtgggtctg aaacgataat ggtgaatatc cctgcctaac tctattcact
     5761 atagaaagta cagcaaaaac tattcttaaa cctaccaagc ctcctactat cattatgaat
     5821 aattttatat accacagcca atttgttatg ttaaaccaat tccacaaact tgcccattta
     5881 tctaattcca ataattcttg ttcattcttt tcttgctggt tttgcgattc ttcaattaag
     5941 gagtgtatta agcttgtgta attgttaatt tctctgtccc actccatcca ggtcgtgtga
     6001 ttccaaatct gttccagaga tttattactc caactagcat tccaaggcac agcagtggtg
     6061 caaatgagtt ttccagagca accccaaatc cccaggagct gttgatcctt taggtatctt
     6121 tccacagcca ggattcttgc ctggagctgc ttgatgcccc agactgtgag ttgcaacaga
     6181 tgctgttgcg cctcaatagc cctcagcaaa ttgttctgct gctgcactat accagacaat
     6241 aattgtctgg cctgtaccgt cagcgtcatt gacgctgcgc ccatagtgct tcctgctgct
     6301 cccaagaacc caaggaacaa agctcctatt cccactgctc ttttttctct ctgcaccact
     6361 cttctctttg ccttggtggg tgctactcct aatggttcaa tttttactac tttatattta
     6421 tataattcac ttctccaatt gtccctcata tctcctcctc caggtctgaa gatcagcggc
     6481 cgcttgctgt gcggtggtct tacttttgtt ttgctcttcc tctatcttgt ctaaagcttc
     6541 cttggtgtct tttatctcta tcctttgatg cacacaatag agggttgcta ctgtattata
     6601 taatgatcta agttcttctg atcctgtctg aagggatggt tgtagctgtc ccagtatttg
     6661 tctacagcct tctgatgttt ctaacaggcc aggattaact gcgaatcgtt ctagctccct
     6721 gcttgcccat actatatgtt ttaatttata ttttttcttt ccccctggcc ttaaccgaat
     6781 tttttcccat cgcgatctaa ttctcccccg cttaatactg acgctctcgc acccatctct
     6841 ctccttctag cctccgctag tcaaaatttt tggcgtactc accagtcgcc gcccctcgcc
     6901 tcttgccgtg cgcgcttcag caagccgagt cctgcgtcga gagagctcct ctggtttccc
     6961 tttcgctttc aagtccctgt tcgggcgcca ctgctagaga ttttccacac tgactaaaag
     7021 ggtctgaggg atctctagtt accagagtca cacaacagac gggcacacac tacttgaagc
     7081 actcaaggca agctttattg aggcttaagc agtgggttcc ctagttagcc agagagctcc
     7141 caggctcaga tctggtctaa ccagagagac ccgtttatgt atcgagctag gcacttaaat
     7201 acaatatctc tgcaatgcgg caattcagtg gttcgtccaa tccatgtcag acccgtctgt
     7261 tgccttccta ataaggcacg atcgtaccac cttacttcca ccaatcggca tgcacggtgc
     7321 tttttctctc cttgtaaggc atgttgctaa ctcatcgtta ccatgttgca agactacaag
     7381 agtattgcat aagactacat taagcttgca gctccagctt ttgttccctt tagtgagggt
     7441 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     7501 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     7561 gagtgagcta actcacctta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     7621 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg ga
//
