LOCUS       sequence-364212-        7198 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Lentiviral construct for expression of mCerulean.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7198)
  AUTHORS   Greiner D, Scott TM, Olson GS, Aderem A, Roh-Johnson M, Johnson JS
  TITLE     Genetic Modification of Primary Human Myeloid Cells to Study Cell 
            Migration, Activation, and Organelle Dynamics.
  JOURNAL   Curr Protoc. 2022 Aug;2(8):e514. doi: 10.1002/cpz1.514.
  PUBMED    36018279
REFERENCE   2  (bases 1 to 7198)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Curr 
            Protoc."; date: "2022-08"; volume: "2(8):e514. doi"; pages: " 
            10.1002/cpz1.514"
COMMENT     Sequence Label: pLKO-cell-mCer
FEATURES             Location/Qualifiers
     source          1..7198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(115..134)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      928..1516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1417..1436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1670..1687
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1804..1825
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1840..1845,1846..1863,1864..1870)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1878..1894
                     /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)."
     primer_bind     1883..1905
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1902..1918
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1902..1918
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1937..1957
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1939..1957
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1985..2211
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2212..2392
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2439..2564
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3057..3290
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3475..3519
                     /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"
     promoter        3817..4065
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3817..3837
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3988..4007
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    4128..4245
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4294..4804
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4723..4741
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(4831..4833,4834..4836,4837..5550)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4876..4897)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5137..5156)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5484..5505
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5701..5934
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6006..6127
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6043..6062)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6097..6116
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6167..6302
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6322..6341)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6323..6341)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6351..6368)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6351..6367)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6360..6382)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6509..6964
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6596..6615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6806..6827
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6990..7094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7095..7163,7164..7198,1..757)
                     /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"
ORIGIN
        1 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
       61 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      121 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      181 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      241 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
      301 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
      361 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
      421 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
      481 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
      541 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
      601 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      661 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      721 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
      781 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
      841 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
      901 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
      961 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1021 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1081 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1141 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1201 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1261 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1321 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1381 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1441 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1501 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1561 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1621 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1681 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     1741 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     1801 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     1861 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     1921 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
     1981 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
     2041 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
     2101 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
     2161 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
     2221 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     2281 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     2341 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
     2401 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
     2461 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
     2521 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
     2581 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
     2641 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
     2701 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
     2761 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     2821 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     2881 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     2941 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     3001 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     3061 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     3121 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     3181 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     3241 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     3301 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     3361 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     3421 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     3481 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     3541 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     3601 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     3661 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
     3721 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtatcga
     3781 tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct
     3841 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     3901 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     3961 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     4021 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggtcc gcaggtatgc
     4081 acgcgtgaat tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa
     4141 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     4201 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     4261 gacagcagag atccactttg gccgcggctc gagggggttg gggttgcgcc ttttccaagg
     4321 cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg
     4381 cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc
     4441 cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc
     4501 cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct
     4561 cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa
     4621 tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc
     4681 ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa
     4741 gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc
     4801 ccagggggat ccgacgtcac cggagttacc atggtgagca agggcgagga gctgttcacc
     4861 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     4921 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     4981 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgagctg gggcgtgcag
     5041 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     5101 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     5161 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     5221 ttcaaggagg acggcaacat cctggggcac aagctggagt acaacgccat ccacggcaac
     5281 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcgg cctcaactgc
     5341 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     5401 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa
     5461 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     5521 actctcggca tggacgagct gtacaagtaa cgtacggata tctgtacaag taacgcccgc
     5581 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg gtacctttaa
     5641 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     5701 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     5761 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     5821 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     5881 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta
     5941 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     6001 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     6061 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     6121 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     6181 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     6241 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     6301 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     6361 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     6421 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     6481 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     6541 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     6601 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     6661 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     6721 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     6781 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     6841 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     6901 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     6961 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     7021 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     7081 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     7141 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctga
//
